Techniques For Phosphopeptide Enrichment Prior To Mass Spec Anal

TECHNIQUES FOR PHOSPHOPEPTIDE ENRICHMENT PRIOR TO ANALYSIS BY MASS SPECTROMETRY
Jamie D. Dunn,1 Gavin E. Reid,1,2 and Merlin L. Bruening1* 1 Department of Chemistry, Michigan State University, East Lansing, Michigan 48824 2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
Received 2 July 2008; received (revised) 17 November 2008; accepted 17 November 2008
Published online 4 March 2009 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20219
Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal afnity chromatography, reversible covalent binding, and metal oxide afnity chromatography. Capture of phosphopeptides on TiO2 seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purication of small (mL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. # 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:2954, 2010 Keywords: phosphorylation; enrichment; IMAC; titanium dioxide; magnetic beads; metal oxides
I. ABBREVIATIONS
ACTH BSA CHAPS a-CHCA 2,5-DHB adrenocorticotropic hormone bovine serum albumin 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate a-cyano-4-hydroxycinnamic acid matrix 2,5-dihydroxybenzoic acid
N,N0 -diisopropylethylamine dithiothreitol N,N0 -dimethylaminopropyl ethyl carbodiimide ethylenediaminetetraacetic acid electrospray ionization Fourier transform ion cyclotron resonance 3-glycidoxypropyltrimethoxysilane glycidyl methacrylate cyclic guanosine monophosphate 2-hydroxyethyl methacrylate b-hydroxypropanoic acid iminodiacetate immobilized metal afnity chromatography isobaric tag for relative and absolute protein quantitation LC liquid chromatography MALDI matrix-assisted laser desorption/ionization MOAC metal oxide afnity chromatography MS mass spectrometry NP nanoparticle NTA nitrilotriacetate PAA poly(acrylic acid) PBS phosphate-buffered saline PEI polyethyleneimine PHEMA poly(2-hydroxyethyl methacrylate) PKG cGMP-dependent kinase PySSPy 2,20 -dithiopyridine SAMs self-assembled monolayers SCX strong cation exchange SALDI surface-assisted laser desorption/ionization SELDI surface-enhanced laser desorption/ionization SILAC stable isotope-labeling of amino acids in cell culture TEOS tetraethyl orthosilicate TFA triuoroacetic acid THAP 20 ,40 ,60 -trihydroxyacetophenone TMSPED N-[3-(trimethoxysilyl)propyl]ethylenediamine TOF time-of-ight DIPEA DTT EDC EDTA ESI FT-ICR GLYMO GMA cGMP HEMA b-HPA IDA IMAC iTRAQ
II. INTRODUCTION
Protein phosphorylation is one of the most important mechanisms to regulate cellular processes (Graves & Krebs, 1999) such as gene expression and membrane transport (Krebs & Beavo, 1979; Krebs, 1983; Hunter, 2000; Pawson & Nash, 2000;
*Correspondence to: Merlin L. Bruening, Department of Chemistry, Michigan State University, East Lansing, MI 48824. E-mail: bruening@chemistry.msu.edu
Mass Spectrometry Reviews, 2010, 29, 29 54 # 2009 by Wiley Periodicals, Inc.
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DUNN, REID, AND BRUENING
Whitmarsh & Davis, 2000; Adams, 2001; Johnson & Lewis, 2001; Simpson, 2003). In a number of regulatory pathways, protein kinases and phosphatases regulate the post-translational phosphorylation status of serine, threonine, and tyrosine residues (Adams, 2001; Johnson & Hunter, 2005), and disruption of these regulatory pathways can sometimes contribute to diseases such as cancer (Cohen, 2001; Lim, 2005). Thus, identication of phosphorylation sites might be vital to develop new pharmaceutical targets and to understand disease states (Fischer & Krebs, 1955; Cohen, 2001; Lim, 2005; Yang et al., 2006; Yu, Issaq, & Veenstra, 2007). Mass spectrometry is currently the method of choice to detect changes in protein phosphorylation and to identify the position of specic phosphorylation events (Mann et al., 2002). However, even with recent advances in mass spectrometry instrumentation, the detection and identication of phosphorylation sites is challenging. Frequently, because both the amount of phosphorylated protein in eukaryotic cells and the degree of phosphorylation are relatively low (Aebersold & Goodlett, 2001; Mann et al., 2002; Simpson, 2003) highly sensitive methods are needed. Additionally, two studies suggested that ionization efciencies and MS signals of phosphorylated peptides are lower than those of their nonphosphorylated analogues, and this factor could make it difcult to detect phosphorylated species in the presence of an abundance of nonphosphorylated peptides (Craig et al., 1994; Liao et al., 1994). For matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), desorption/ionization efciencies for phosphopeptides were reported to be an order of magnitude lower than those of their nonphosphorylated counterparts, and ionization of phosphorylated peptides can become more difcult as the number of phosphorylation sites increases. More recently, the validity of these results was tested with high-performance LC-MS with a variety of synthetic phosphopeptides and their nonphosphorylated counterparts spiked in tryptic protein digests (Steen et al., 2006). This study concluded that the difculty to detect phosphopeptides with LC-MS stems from the low abundance of the species, not low ionization efciencies. However, the synthetic peptides examined contained many basic amino acid residues such as arginine (Steen et al., 2006), which generally allow for better MS detection (Krause, Wenschuh, & Jungblut, 1999; Clipston, Jai-nhuknan, & Cassady, 2003). Additionally, the peptides were separated with LC prior to analysis by MS, so further studies with mixtures of peptides might be needed. In any case, the detection and identication of phosphorylated species is challenging because of low abundances, so enrichment techniques are typically employed prior to analysis. This article reviews recently developed methods for the selective capture and elution of phosphopeptides prior to analysis, along with adaptations of these methods for both enrichment using magnetic beads and on-plate purication prior to MALDI-MS. Although there are numerous reports on the application of mass spectrometry for identifying phosphorylation sites, the focus here is on enrichment techniques, not applications. The three major methods to capture phosphopeptides include immobilized metal afnity chromatography (IMAC), reversible covalent binding, and metal oxide afnity chromatography (MOAC), and these methods are discussed in Section II. Avidin afnity chromatography has also been described for
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phosphopeptide capture (Adamczyk, Gebler, & Wu, 2001; Oda, Nagasu, & Chait, 2001; Goshe et al., 2002), but this technique requires a number of steps and relatively large sample quantities and is not described here. Strong cation exchange (SCX) chromatography is another recent alternative employed for phosphopeptide enrichment, (Ballif et al., 2004; Beausoleil et al., 2004; Lim & Kassel, 2006; Olsen et al., 2006; Trinidad et al., 2006; Wu et al., 2007) but this technique has also been reviewed recently (Gafken & Lampe, 2006; Yu, Issaq, & Veenstra, 2007). SCX is frequently employed in combination with IMAC, MOAC, and covalent techniques. Finally, immunoprecipitation is capable of enriching tyrosine-phosphorylated proteins and peptides and greatly aids in the identication of sites of tyrosine phosphorylation (Zhang & Neubert, 2006; Schmidt, Schweikart, & Andersson, 2007). In hyperphosphorylated Jurkat cells, Rush and co-workers used phosphotyrosine-specic antibodies to enrich tryptic tyrosine phosphopeptides and identify 194 phosphotyrosine sites (Rush et al., 2005). Some studies employed antibodies to specic motifs in phosphothreonine and phosphoserine peptides (Grnborg et al., 2002; Matsuoka et al., 2007), but antibody-based isolation of phosphoserine and phosphothreonine peptides is not yet a general technique (Kristjansdottir et al., 2008; Sopko & Andrews, 2008) so this method is not discussed here. Because recent reviews focused on IMAC and covalent capture (Reinders & Sickmann, 2005; Morandell et al., 2006; Schmelzle & White, 2006; DAmbrosio et al., 2007; Yu, Issaq, & Veenstra, 2007), we will present these methods relatively briey and will describe advances in MOAC in more detail. Section III of this review describes the adaptation of IMAC and MOAC for selective capture on magnetic beads and isolation of phosphopeptides directly on MALDI plates. Although these platforms may not be well-suited to identify all of the phosphopeptides in a complex sample such as a cell extract, they might prove useful to rapidly analyze small volumes of dilute samples. Finally, Section IV presents a brief perspective on phosphopeptide enrichment.
III. PHOSPHOPEPTIDE CAPTURE AND ELUTION A. Immobilized Metal Afnity Chromatography

Immobilized metal afnity chromatography (IMAC) relies on the interactions between metal-ligand complexes and specic functional groups such as phosphates. The enrichment procedure includes selective binding to the resin, a rinse to remove impurities, and elution to yield a concentrated solution of the species of interest, as shown in Figure 1. Although IMAC is less specic than other afnity methods, this lower specicity makes it applicable to a wide range of separations, including enrichment of phosphorylated species. IMAC was introduced in 1975 (Porath et al., 1975), and has historically been the method most commonly used to isolate phosphopeptides prior to analysis by MS (Neville et al., 1997; Li & Dass, 1999). Iminodiacetate (IDA) and nitrilotriacetate (NTA), shown in Figure 2, are the prototypical metal-binding ligands employed in IMAC stationary phases. NTA was introduced to IMAC in 1987 (Hochuli, Dobeli, & Schacher, 1987), and the Fe(III)NTA
Mass Spectrometry Reviews DOI 10.1002/mas
TECHNIQUES FOR PHOSPHOPEPTIDE ENRICHMENT
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FIGURE 1. Schematic diagram of phosphopeptide isolation by IMAC or MOAC. The column packing is different for the two techniques. [Color gure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
complex is probably the complex most frequently employed to enrich phosphopeptides, although Ga(III), Zr(IV), and Al(III) complexes have also been investigated for their afnity towards phosphorylated species (Posewitz & Tempst, 1999; Nuhse et al., 2003). Alternative metal-ligand complexes of Zr(IV)-phosphonate immobilized on various stationary phases or supports have been recently employed for phosphopeptide enrichment by several groups (Zhou et al., 2006; Dong et al., 2007; Feng et al., 2007; Yu, Issaq, & Veenstra, 2007; Wei et al., 2008).
FIGURE 2. Structures of (a) IDA and (b) NTA.
There are a number of challenges associated with using IMAC. First, because the metal ions are not covalently bound to the substrate, there is a possibility to leach these ions from the column during the enrichment steps, which might lead to loss of phosphopeptides or to a contamination of peptides with metal ions. However, thorough washing of the column before use and a judicious selection of binding ligands can minimize these problems. For example, IDA is a tridentate ligand, whereas NTA is a tetradentate ligand; hence, the latter binds more strongly to the metal ion to better prevent leaching (Hochuli, Dobeli, & Schacher, 1987; Holmes & Schiller, 1997). A second challenge is nonspecic binding of peptides that contain the acidic residues glutamic and aspartic acid. A few reports suggested that increasing the ionic strength of the loading or a rinsing solution can help minimize electrostatic interactions between acidic residues and metal-ion complexes (Andersson & Porath, 1986; Holmes & Schiller, 1997; Kagedal, 1998; Ficarro et al., 2002). However, another study did not see a signicant reduction in nonspecic adsorption by incorporating salts into the enrichment protocol (Ndassa et al., 2006). To better overcome this unwanted binding, the carboxyl groups of amino acid residues can be converted to methyl esters (Ficarro et al., 2002). Peptides are typically esteried by reaction with acetyl chloride
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in an excess of methanol (methanolic HCl). More recently, thionyl chloride in methanol was used for formation of the methyl esters of phosphopeptides (Moser & White, 2006; Ndassa et al., 2006). The authors reported that this reagent showed a higher efciency for the conversion of carboxylic acid groups to methyl esters than did methanolic HCl. Unfortunately, incomplete esterication of phosphopeptides will increase chemical noise, and sample losses during lyophilization of the esteried peptides may be around 20% (Stewart, Thomson, & Figeys, 2001). Bodenmiller and co-workers examined the effect of methyl esterication on the identication of phosphopeptides that were isolated from a cytosolic fraction of Drosophila melanogaster Kc167 cells (Bodenmiller et al., 2007a). Methyl esterication prior to IMAC allowed identication of 199 peptides, whereas IMAC prior to methyl esterication permitted identication of 193 peptides. Interestingly, the overlap of the peptides identied by the two protocols was only approximately 30%. Nonspecic binding to Ga(III)-IMAC resins can also be reduced by using the endoproteinase glu-C rather than trypsin for protein digestion (Seeley, Riggs, & Regnier, 2005). Glu-C cleaves the protein at the C-terminus of glutamic and aspartic acid residues so that only one acidic residue will be present in the peptide chains. Hence, glu-C digestion should alleviate essentially all nonspecic binding due to interactions between IMAC resins and multiple acidic residues. Another challenge that has been reported in the use of IMAC is that the technique is more specic for multiply phosphorylated peptides than monophosphorylated peptides because multiply phosphorylated peptides bind more strongly to the IMAC resin (Ficarro et al., 2002; Nuhse et al., 2003; Nousiainen et al., 2006; Jensen & Larsen, 2007). Thingholm and co-workers utilized the strong binding of multiply phosphorylated peptides to IMAC resins to increase the number of phosphorylated peptides detected from lysates of human mesenchymal stem cells (Thingholm et al., 2008). They rst eluted monophosphopeptides from the IMAC column using 1% TFA and puried both the column ow-through and the eluent a second time using TiO2. Elution using NH4OH then yielded a fraction rich in multiply phosphorylated peptides. Overall, this combination of enrichment techniques facilitated identication of 492 phosphorylated peptides, including 186 multiply phosphorylated peptides, whereas the use of just TiO2 enrichment revealed only 286 phosphopeptides, 54 of which were multiply phosphorylated. Thus, the combination of IMAC with different eluents is particularly useful for detecting multiply phosphorylated species. The specicity of IMAC for binding multiply phosphorylated peptides depends on variables such as the afnity ligand, the binding capacity of the support material, and the binding, rinsing, and elution conditions. Recently, the use of a high-capacity IMAC material in conjunction with an optimized binding and rinsing buffer (33:33:33 acetonitrile/methanol/aqueous 0.1% acetic acid) led to enhanced phosphopeptide recovery and uniform LC-MS detection of multiply and singly phosphorylated peptides (Ndassa et al., 2006). Peptides that contained acid residues were converted to methyl esters in this study. If the IMAC column does not have a large enough capacity for all of the phosphorylated peptides in the mixture, then the multiply phosphorylated peptides will bind preferentially over the mono32
phosphorylated species. Reducing the concentration of acetic acid from 1% to 0.1% yielded an increase in the number of phosphorylated peptides detected, and the recovery of phosphorylated peptides from a cell lysate doubled with 33:33:33 acetonitrile/methanol/aqueous 0.1% acetic acid as the loading and washing solutions instead of 25:75 acetonitrile/ aqueous 1% acetic acid that contained 100 mM NaCl. In other experiments, the use of 100 mM NaCl had the greatest effect on monophosphorylated peptide recoveries, which decreased by half when NaCl was present. Another group implemented triuoroacetic acid (TFA) rather than acetic acid, and a high content of acetonitrile in the loading and rinsing solutions to reduce nonspecic adsorption from acidic and hydrophobic peptides (Kokubu et al., 2005). Effective loading and washing solutions for a pipette tip loaded with IMAC resin (Phos-Select gel from Sigma, St. Louis, MO) contained 4060% acetonitrile and 0.11% TFA. In some cases, IMAC columns were applied to relatively complex samples. Giorgianni and co-workers used IMAC along with both a C18 column and a POROS Oligo R3 column to collect phosphopeptide fractions from prostate cancer cells (Giorgianni et al., 2007). This study identied 137 phosphorylation sites in 81 phosphoproteins. Related work used a combination of isoelectric focusing and IMAC to identify 50 phosphorylation sites in 26 proteins obtained from primary pituitary tissue (BeranovaGiorgianni et al., 2006). Thus, IMAC can be applied to relatively complex samples, but it is typically used in combination with other separation techniques. As mentioned above, the use of multiple fractions from IMAC allowed identication of 492 phosphopeptides in human stem cells (Thingholm et al., 2008).
B. Reversible Covalent Binding

In principle, covalent binding might offer higher enrichment selectivities than IMAC, but typical covalent procedures frequently employ a number of steps. In 2001, a multistep, solid-phase enrichment technique that can be applied to phosphotyrosine peptides in addition to phosphoserine- and phosphothreonine-containing peptides was developed (Zhou, Watts, & Aebersold, 2001). The method is portrayed in Figure 3. Prior to tryptic digestion, any cysteine residues of the protein mixture are reduced and alkylated. After digestion, the amino groups of the peptides are rst protected with t-butyl-dicarbonate (tBoc), and the carboxylate and phosphate groups are converted to amides and phosphoramides through a carbodiimide (N,N0 -dimethylaminopropyl ethyl carbodiimide, EDC)-mediated reaction with ethanolamine. The phosphate group is regenerated by acid hydrolysis (10% TFA), and cystamine is attached to the phosphate group via a carbodiimide-catalyzed condensation. Finally, the attached cystamine is reduced with dithiothreitol (DTT), and the free sulfhydryl group is reacted with iodoacetyl groups immobilized on glass beads. After rinsing the beads with 2 M NaCl, methanol, and water to remove nonphosphorylated peptides, the phosphopeptides are cleaved from the surface and the tBoc protecting groups are removed with 100% TFA. Using this elaborate enrichment strategy for the analysis of the phosphotyrosine peptide TTHpYGSLPQK in a
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FIGURE 3. Solid-phase enrichment using carbodiimide condensation and glass beads derivatized with iodoacetyl groups. In the nal step, the phosphopeptide is cleaved from the bead and the tBoc protecting group is removed using 100% TFA, which is not shown in this gure (procedure from Zhou, Watts, & Aebersold, 2001).
digest of phosphorylated myelin basic protein, recovery was only 20% (Zhou, Watts, & Aebersold, 2001). b-Elimination chemistry provides a considerably simpler method to enrich phosphopeptides. In this chemistry, strong bases such as NaOH or Ba(OH)2 are used to cleave the phosphoester bonds of phosphoserine and phosphothreonine to form the respective dehydroalanine or dehydroaminobutyric acid analogs, which can react with different nucleophiles, such as thiol, amine, or alcohol groups (Fig. 4). These reactions were
used to cleave the phosphate ester of a-casein phosphopeptides from a tryptic digest (cysteines were oxidized to cysteic acid prior to digestion), and these peptides were subsequently reacted with propanedithiol and covalently bound to a solid support derivatized with reactive dithiopyridine groups (Thaler et al., 2003). After the resin was rinsed thoroughly, the bound peptides were simply cleaved using DTT, and the free thiol groups were alkylated. The sample was desalted and analyzed using MALDITOF-MS. However, when combined with MALDI-MS, this
FIGURE 4. Reversible solid-phase enrichment of phosphoserine-containing peptides using b-elimination and Michael addition followed by enrichment on a dithiopyridino-modied resin (pro edure from Thaler et al., 2003). Enrichment can also be applied to phosphothreonine-containing peptides.
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method revealed only 2 tryptic phosphopeptides from a 20-mg ($820 pmol) a-casein digest. a-Casein contains a-S1 and a-S2 protein forms, and complete tryptic digestion of these proteins should give a total of 9 phosphopeptides. Another covalent enrichment technique employed an adiazo functionalized resin to reversibly and covalently bind the phosphate group of phosphopeptides (Lansdell & Tepe, 2004). Because b-elimination or another technique is not required to chemically derivatize the phosphopeptide prior to solid-phase enrichment, this technique can be applied to phosphorylated serine, threonine, and tyrosine peptides. Figure 5 shows the immobilization procedure. To prevent carboxylate groups of the peptides from covalently binding to the resin, these groups were rst converted to methyl esters. The authors used the a-diazo functionalized resin to enrich 500 fmol of phosphorylated angiotensin II (DRVpYIHPF) from a mixture of three nonphosphorylated peptides. After the peptide mixture was incubated with the a-diazo resin, the nonphosphopeptides were rinsed away, and the immobilized phosphopeptide was cleaved with either TFA or NH4OH. The resulting peptides were analyzed with MALDI-TOF-MS, and the mass spectrum showed a single peak, which was due to phosphorylated angiotensin (m/z 1127).
Figure 6 shows the immobilization of methyl-esteried phosphopeptides onto an amino-terminated dendrimer with EDC coupling (Tao et al., 2005). This one-pot chemistry avoids the need to protect the amino groups on the N-terminus and lysine or arginine residues, presumably because of the large excess of amine groups on the dendrimers. Once the phosphoprotein digest had incubated with the dendrimer for 10 hr, the dendrimer was rinsed with 3 M NaCl, 30% methanol, and water, and the phosphopeptides were liberated (10% TFA, 30 min) to cleave the phosphoramidate bonds. This technique was employed to analyze digests of b-casein, and the phosphopeptide recovery was greater than 35%. The same group also showed that phosphopeptides activated with EDC also react with excess cystamine to form phosphoramidate bonds. The modied phosphopeptides can then be immobilized via reaction of reduced cystamine thiol groups with maleimide functionalities in pore glass. Finally, cleavage of the phosphoramidate bond under acidic conditions liberates the peptide. Using this method, the recovery of 100 fmol of phosphorylated angiotensin from a 500 pmol BSA digest was approximately 40% (Bodenmiller et al., 2007b). Warthaka and co-workers developed a reversible solidphase enrichment technique based on oxidation-reduction
FIGURE 5. Reversible solid-phase enrichment using an a-diazo resin (Lansdell & Tepe, 2004). Cleavage of
the phosphopeptide can be accomplished by using either triuoroacetic acid (TFA) or NH4OH. Note that when NH4OH is used, the methyl ester is hydrolyzed back to the original phosphopeptide.
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FIGURE 6. Three-step solid phase enrichment using carbodiimide condensation (procedure from Tao
et al., 2005).
condensation of phosphopeptides with glycine-derivatized Wang resin as shown in Figure 7 (Warthaka, KarwowskaDesaulniers, & Pum, 2006). In the rst step, carboxylic acidcontaining residues were protected via methyl esterication. Next, the methylated phosphopeptides were covalently coupled to the glycine Wang resin in the presence of triphenylphosphine (PPh3), 2,20 -dithiopyridine (PySSPy), and N,N0 -diisopropylethylamine (DIPEA). The phosphopeptide was bound to the glycine-derivatized resin via a phosphoramidate bond, which is cleavable with TFA. Subsequently, the resin was washed and the phosphopeptides were eluted with 95% TFA and analyzed with MALDI-TOF-MS. By using this
enrichment strategy, the recovery of a monophosphopeptide from a b-casein digest was $37% (Warthaka, KarwowskaDesaulniers, & Pum, 2006), which was comparable to that of the carbodiimide solid-phase enrichment technique mentioned above (Tao et al., 2005).
C. Metal Oxide Afnity Chromatography

The use of MOAC for phosphopeptide enrichment has grown rapidly in the past 4 years because of high recoveries and selectivities, even without methyl esterication of peptides.
FIGURE 7. Reversible solid-phase enrichment of protected phosphopeptides using an oxidation-reduction condensation reaction (procedure from Warthaka, Karwowska-Desaulniers, & Pum, 2006). The amino terminus was acetylated using acetic anhydride.
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Additionally, the metal oxides are often more stable than traditional silica-based stationary phases. ZrO2, for example, is stable at temperatures up to 2008C and over a pH range from 1 to 14 (Nawrocki et al., 2004). Metal oxides that have been applied towards selective phosphopeptide enrichment include titanium dioxide (TiO2), zirconium dioxide (ZrO2), aluminum oxide (Al2O3), and niobium oxide (Nb2O5). Aluminum hydroxide has also been used. Below we discuss phosphopeptide enrichment studies with each of these materials.
1. Enrichment with Titanium Dioxide

Currently, titanium dioxide is the most popular metal oxide resin used as a selective afnity support to capture phosphorylated compounds, including peptides (Ikeguchi & Nakamura, 1997, 2000; Pinkse et al., 2004; Sano & Nakamura, 2004a,b; Larsen et al., 2005). At acidic pH, TiO2 has a positively charged surface (Kosmulski, 2002) that selectively adsorbs phosphorylated species and exhibits outstanding enrichment behavior for phosphopeptides (Fig. 1). Notably, when coupled with appropriate solutions for sample loading, column rinsing, and peptide elution, TiO2 is highly selective to preferentially bind phosphopeptides over acidic peptides (Larsen et al., 2005; Thingholm et al., 2006; Jensen & Larsen, 2007; Sugiyama et al., 2007). TiO2 precolumns and 2D-nano-LC-ESI-MS/MS were utilized to analyze a synthetic phosphopeptide as well as proteolytic digests of cGMP-dependent protein kinase (Pinkse et al., 2004). The protocol called for a phosphopeptide loading solution in 0.1 M acetic acid, a rinsing solution of 0.1 M acetic acid in 80% acetonitrile, and an ammonium bicarbonate eluent (pH 9.0). When an equimolar mixture of 125 fmol of RKIpSASEF, a synthetic phosphorylated peptide derived from cGMP-dependent protein kinase (PKG), and 125 fmol of its nonphosphorylated counterpart were enriched with the TiO2 precolumn, the percent recovery of the RKIpSASEF was above 90%. Additionally, 11 phosphorylated peptides were recovered from the analysis of a PKG tryptic digest. However, nonphosphorylated peptides were also retained on the titanium dioxide precolumn. The authors thought that the acidic nature of these nonphosphorylated peptides caused them to have an afnity for TiO2, so they compared the afnity of the methylated and nonmethylated forms of [Glu1]-brinopeptide B (EGVNDNEEGFFSAR) for titania. Their studies showed that 98% of the nonmethylated peptide was retained on the TiO2 precolumn under the phosphopeptide loading and washing procedure, and eluted under basic conditions. In contrast, the methylated form appeared to have very little afnity for TiO2. In other developments with TiO2, the modication of the loading buffer and rinsing solution with 0.1% TFA and 2,5dihydroxybenzoic acid (2,5-DHB) rather than 0.1 M acetic acid improved the detection of phosphorylated peptides (Larsen et al., 2005). Hart and co-workers rst introduced 2,5-DHB as an eluent for IMAC (Hart et al., 2002). The pH values of 0.1% TFA and 0.1 M acetic acid solutions are 1.9 and 2.7, respectively; hence, the TFA solution protonates acidic residues and prevents adsorption of nonphosphorylated peptides to TiO2. Moreover, an NH4OH eluent at pH 10.5 afforded a high recovery of phosphopeptides. The pKa and pKb values for titania are 4.4 and
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7.7, respectively (Koizumi & Taya, 2002), so at high pH, the titania should become negatively charged to allow phosphopeptide elution. (The rst and second pKa values of phosphoserine and phosphothreonine are <1.7 and 6, respectively; Vogel, 1989; Xie, Jiang, & Ben-Amotz, 2005.) Four additional phosphopeptides were observed from an a-casein digest with NH4OH rather than pH 9.0 ammonium bicarbonate as the eluent. Remarkably, when 2,5-DHB was used in the binding and rinsing solution and NH4OH, pH 10.5 was the eluent, 20 phosphorylated peptides were detected in the MALDI mass spectrum of a 500 fmol a-casein digest, whereas virtually no signals due to nonphosphorylated peptides were observed (Larsen et al., 2005). Larsen and co-workers also optimized conditions when they analyzed a more complex digest mixture that contained equimolar amounts (500 fmol) of three nonphosphorylated proteins (bovine serum albumin (BSA), b-lactoglobulin, and carbonic anhydrase) and three phosphoproteins (b-casein, a-casein, and ovalbumin). Using enrichment with TiO2 along with the loading solutions and eluents mentioned above, they were able to recover 18 phosphorylated peptides, whereas the majority of peaks due to nonphosphorylated peptides were virtually eliminated. It should be noted that after the TiO2 column, these studies typically used a Poros Oligo R3 microcolumn for sample desalting and concentration. Occasionally, phosphopeptides were not retained on the Oligo material and required further purication on a graphite microcolumn. An enrichment comparison was made between this titania material and Fe(III)NTAIMAC beads (PHOS-select, Sigma) with a mixture of the digested proteins described above. At 10:1 and 50:1 molar ratios of nonphosphoproteins to phosphoproteins in tryptic digests, the use of the commercial PHOS-select IMAC beads (Sigma) yielded fewer phosphorylated peptide signals and more peaks due to nonphosphopeptides compared to results obtained with the TiO2 resin (Larsen et al., 2005). The IMAC loading and rinsing solution consisted of 250 mM acetic acid, 30% acetonitrile, and the eluent was NH4OH, pH 10.5. This study also compared the use of MALDI-MS and LC-ESI-MS/MS for phosphopeptide analysis. LC-ESI-MS/MS analysis resulted in the detection of eight phosphopeptides, whereas MALDI-MS detected >16. Many phosphopeptides that were not detected with LC-ESI-MS/MS were multiply phosphorylated. A similar result was also observed in another study (Gruhler et al., 2005). In addition to examining 2,5-DHB in loading solutions, the competitive effects of other acids (some structures are shown in Fig. 8) on the binding of nonphosphorylated peptides to TiO2 were also examined (Larsen et al., 2005). These studies gave the following order of ability to inhibit nonphosphopeptide binding: 2,5-DHB $ salicylic acid $ phthalic acid > benzoic acid $ cyclohexane carboxylic acid > phosphoric acid > TFA > acetic acid. IR spectroscopy showed that substituted aromatic carboxylic acids interact more strongly with TiO2 than do aliphatic carboxylic acids that contain one COOH group (Dobson & McQuillan, 2000). Phosphoric acid (Langmuir binding constant of 4 104 M1 at pH 2.3) and substituted aromatic carboxylic acids (binding constants of 104 105 M1) have similar binding afnities for TiO2 (Connor & McQuillan, 1999), but the coordination geometry of salicylate and phosphate to TiO2 are different. Salicylic acid creates a chelating bidentate structure with the TiO2 surface, whereas a bridging bidentate complex
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FIGURE 8. Structures of acids used in an attempt to minimize nonspecic binding to TiO2.
forms when phosphate (from phosphoric acid) binds to the surface. Larsen et al. suggested that due to such differences in coordination geometry, 2,5-DHB predominantly competes for binding sites with nonphosphorylated peptides and not phosphorylated peptides (Larsen et al., 2005). Thingholm and co-workers recently provided a protocol to enrich phosphopeptides ofine with titania-packed pipette tips prior to analysis with either LC-MS or MALDI-MS (Thingholm et al., 2006). Some of the main enrichment conditions included a loading solution that contained 100300 mg/mL of 2,5-DHB in 80% acetonitrile, 2% TFA (5% TFA was recommended for more complex samples), a washing solution comprised of 80% acetonitrile and 2% TFA, and an elution solution of 0.5% NH4OH (recommended pH should be !10.5). Additionally, the protocol recommended desalting of an acidied phosphopeptide eluent with POROS Oligo R3, a resin designed for phosphodiester and phosphorothioate oligonucleotide purication (Applied Biosystems, http://www3.appliedbiosystems.com/ AB_Home/index.htm, May 6, 2008). For the analysis of enriched phosphopeptides with MALDI-MS, the suggested matrix was 20 mg/mL of 2,5-DHB in 50% acetonitrile, 1% H3PO4. Similar enrichment methods (no ofine eluent desalting step was performed prior to MS analysis) and analysis with MALDIMS/MS and nano-LC-ESI-MS/MS were used to identify new phosphorylation sites in proteins isolated from spinach stroma membranes (Rinalducci et al., 2006). Strong cation exchange (SCX) and titanium dioxide chromatography were utilized for phosphopeptide enrichment and stable-isotope labeling by amino acids in cell culture (SILAC) for quantitation to study phosphorylation changes upon in vivo stimulation of HeLa cells with epidermal growth factor (Olsen et al., 2006). First, fractions of digested HeLa cell extracts were collected from SCX chromatography, and the phosphopeptides were further enriched with TiO2-packed tips with 2,5-DHB in the loading solution (rinse solutions consisted of either 10% or 80% acetonitrile, 0.1% TFA, and elution solutions contained either 20% or 40% acetonitrile with NH4OH, pH 10.5). The enriched fractions were dried, and the residue was reconstituted in 5% acetonitrile, 0.1% TFA, and subsequently was analyzed with nano-LC-ESI-MS (multistage MS was used to characterize the phosphopeptides). This protocol resulted in the detection of over 10,000 phosphopeptides from over 2,200 proteins. Interestingly, this study showed that the relative abundance of phosphorylation sites of tyrosine, threonine, and serine, based on more than 2,000 proteins, was 1.8%, 11.8%, and 86.4%, respectively. This result may be a better representation of the abundance of tyrosine phosphorylation than the value of $0.05% found from phosphoamino acid analysis (Hunter & Sefton,
1980). However, we should note that the mass spectrometry study was performed with serum-starved cells, which might change the relative abundances of the phosphorylated amino acids. Although the incorporation of 2,5-DHB in binding and/or rinsing solutions minimizes nonspecic adsorption of acidic nonphosphorylated peptides without hindering phosphopeptide binding to TiO2 (Larsen et al., 2005), a few recent studies suggested alternative compounds as nonphosphopeptide excluders that minimize adsorption of nonphosphopeptides (Jensen & Larsen, 2007; Sugiyama et al., 2007; Yu et al., 2007). One of the main reasons to examine new excluders is that 2,5DHB is not highly compatible with LC-ESI-MS because of column clogging, loss of sensitivity over time, ion suppression, and coelution of 2,5-DHB with phosphopeptides (Sugiyama et al., 2007). The incorporation of 300 mg/mL of lactic acid in the loading and washing solution enabled the recovery of 12 phosphopeptides from a digest mixture of a-casein, fetuin, and phosvitin (2.5-mg of each protein) with TiO2 enrichment. Importantly, only phosphopeptides were detected. In comparison, when 300 mg/mL of 2,5-DHB, rather than lactic acid, was used, three nonphosphopeptides and only four phosphopeptides were observed (Sugiyama et al., 2007). When the lactic acid protocol was applied to a 2.5-mg phosphoprotein digest mixture containing a-S1-casein, a-S2-casein, fetuin, and phosvitin, the average recovery of 13 phosphopeptides was approximately 50%. In experiments with more complex samples, Sugiyama et al. identied 1100 phosphopeptides from HeLa cells using enrichment on TiO2 with lactic acid as an excluder in the loading and rinsing solutions. When a 3-mg digest of b-casein was applied to a microcolumn that contained 5-mm diameter TiO2 particles, and when 200 mM ammonium glutamate was incorporated into the rinsing solution, 84% phosphopeptide recovery was obtained (Yu et al., 2007). This high recovery shows that glutamate does not displace phosphate from the surface, even though it is a diacid. As suggested earlier, the geometry of some binding sites may be specic for phosphate groups. A rinsing solution with 130 mM 2,5-DHB also afforded high (70%) recovery. The use of the ammonium glutamate as an excluder in the analysis of HeLa cell extracts resulted in identication of 858 phosphopeptides, of which 79% were monophosphorylated peptides and the other 21% were multiply phosphorylated (Yu et al., 2007). These percentages agree with a simulation performed with the Phospho.ELM database version 4.0 (Diella et al., 2004). It was estimated that monophosphopeptides make up roughly 80% of the tryptic phosphorylated peptides of biological samples (Ndassa et al., 2006).
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Lastly, a series of excluders in the loading/washing solutions was evaluated for titanium dioxide afnity chromatography prior to MALDI-MS. These studies demonstrated that compared to conventional IMAC, TiO2 is more compatible with a number of reagents commonly used in the solubilization and digestion of proteins (Jensen & Larsen, 2007). The sample analyzed was a desalted tryptic digest of 12 proteins (250 fmol of each protein), and excluders examined included phthalic, glycolic, oxalic, lactic, gallic, and citric acids. Among these compounds, 1 M glycolic acid in the loading/washing solution, which also contained 5% TFA and 80% acetonitrile, provided the highest phosphopeptide and lowest nonphosphopeptide recoveries (23 phosphopeptides and only two nonphosphopeptides were detected) with TiO2. Glycolic acid, which is the smallest a-hydroxy carboxylic acid, should form a surface chelate that is similar to the proposed chelate with 2,5-DHB, and this may account for its effectiveness as an excluder. Many reagents, such as detergents, surfactants, and salts (with the exception of CHAPS and DMSO) as well as buffer reagents such as EDTA and PBS did not hinder phosphopeptide binding to the TiO2 (Jensen & Larsen, 2007). Overall, the best Fe(III)NTAIMAC recovery of phosphopeptides (18 phosphopeptides and 1 nonphosphopeptide) was obtained when the loading solution contained 0.1% TFA, 50% acetonitrile, and 0.2% RapiGest (surfactant for denaturing protein prior to digestion). However, the number of phosphorylated peptides that bound to the IMAC resin was reduced (compared to results with RapiGest) in the presence of 1% CHAPS (67% reduction), 10 mM EDTA (94% reduction), and PBS (44% reduction). When chromatographic enrichments with ZrO2 and TiO2 were compared, MALDI-MS signals due to multiply phosphorylated peptides were higher and more numerous with TiO2 rather than ZrO2 (Jensen & Larsen, 2007). Monophosphorylated peptides did not have a higher afnity for ZrO2 as reported previously (see below) (Kweon & Hakansson, 2006). Because surface properties of metal oxides play a critical role in the binding of phosphopeptides, a few studies attempted to determine how physical characteristics (particle size and morphology) inuence phosphopeptide adsorption. Nanoparticles might isolate phosphopeptides especially efciently because of their high surface area-to-volume ratio. Crosslinked TiO2 nanoparticles had a twofold higher phosphopeptide binding capacity ($300 mmol of phenyl phosphate per gram of crosslinked TiO2 nanoparticles) than 5-mm diameter TiO2 particles (Liang et al., 2006). These nanoparticles afforded a phosphopeptide recovery of $70% and phosphopeptide MS signals (from casein digests) that were at least twofold higher than those obtained with the use of larger particles. The nanoparticle stationary phase was crosslinked to minimize loss of the particles during the rinsing and elution steps. The above-mentioned studies with titania for phosphopeptide enrichment were carried out ofine, and the samples were analyzed with either LC-MS or MALDI-MS. Recently, optimization of conditions for online TiO2-based enrichment of four phosphopeptides from a tryptic digest of the two casein proteins (1 pmol each) was described (Cantin et al., 2007). The overall optimum conditions for these four peptides included a loading buffer consisting of 20% acetonitrile and 2% formic acid,
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a wash solution comprised of 80% acetonitrile and 2% formic acid, and an elution solution of 200 mM NH4HCO3. For a more complex digest mixture, consisting of an in vitro kinase activated protein and its nonactivated form, the optimum binding buffer was essentially the same, having an acetonitrile content of 520% and 2% formic acid.
2. Enrichment with Zirconium Dioxide

Like titanium dioxide, zirconium dioxide is positively charged at acidic pH and has a higher binding afnity towards phosphate than carboxylate anions (Blackwell & Carr, 1991a,b). The isoelectric points of TiO2 and ZrO2 are $6 and $7, respectively (Vassileva, Proinova, & Hadjiivanov, 1996; Renger et al., 2006). Moreover, ZrO2 has been used previously as a chromatographic resin due to its physical and thermal stability, and thus it is a promising material for phosphopeptide enrichment. In 2006, microtips that contained zirconium dioxide (Glygen, Columbia, MD) were utilized for the enrichment of phosphorylated peptides, and the specicity and recovery achieved was compared to that obtained with Glygen microtips containing titanium dioxide (with the same binding, rinsing and elution solutions) (Kweon & Hakansson, 2006). With microtips packed with 50-mg ZrO2, the optimal procedure for the enrichment of 100-pmol tryptic a-casein and b-casein digests prior to analysis with negative-ion ESI-FT-ICR MS employed a 3.3% formic acid (pH 2.0) binding solution, a water rinse, and a 0.5% piperidine (pH 11.5) elution solution. Under these conditions, ten a-casein phosphorylated peptides and only one nonphosphorylated peptide were detected with the ZrO2 tips, whereas eight phosphopeptides and one nonphosphorylated peptide were detected with the TiO2 tips. Similarly, when a b-casein digest was analyzed with ZrO2 tips, ve phosphopeptides and two nonphosphopeptides were detected, whereas use of TiO2 allowed detection of only four phosphopeptides and at least one nonphosphopeptide. Overall, the TiO2 microtips were more selective for enrichment of multiply phosphorylated peptides, whereas the ZrO2 tips enriched primarily monophosphorylated peptides. The authors suggested that the selectivity of ZrO2 for monophosphorylated peptides could be due to either the higher acidity of zirconia or the higher coordination number compared to titania. However, the surface properties of these metal oxide materials are not well understood. The enrichments of phosphopeptides from tryptic and glu-C digests were also compared with metal oxide resins in pipette tips (Kweon & Hakansson, 2006). As mentioned above, complete glu-C digestion results in only one carboxylate group per peptide and eliminates the problem of unwanted, strong binding by species that contain multiple carboxylate groups (Seeley, Riggs, & Regnier, 2005). However, glu-C digestion did not provide any signicant advantage over tryptic digestion in the enrichment of casein phosphopeptides with either ZrO2 or TiO2 tips. The use of b-hydroxypropanoic acid (b-HPA) as an excluder in the loading solution improved the detection of phosphopeptides with nano-LC-ESI-MS after enrichment on ZrO2 (Sugiyama et al., 2007). Eleven peptides were detected from a 2.5-mg sample of a-casein, fetuin, and phosvitin, whereas signals from nonphosphorylated peptides were not detected. Other
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excluders examined in that study included 2,5-DHB, glycolic acid, lactic acid, malic acid, and tartaric acid. Application of enrichment with ZrO2 and b-HPA to the analysis of proteins digested from HeLa cells allowed detection of 1181 phosphorylated peptides, which is slightly higher than results obtained when titania tips were used (1100 phosphopeptides detected). In another study of ZrO2-based enrichment, 100 mM diammonium hydrogen phosphate (pH 9) was used as the elution solution for the analysis of casein peptides (digested proteins included a-S1-, a-S2-, b-, and k-casein isolated from milk, and most of the peptides resulted from nonspecic cleavage due to milk proteases) (Cuccurullo et al., 2007). After peptides were eluted, they were acidied and analyzed with either surfaceenhanced laser desorption/ionization (SELDI)-MS/MS (normal phase ProteinChip used for sample desalting) or nano-LC-ESIMS/MS. Four phosphorylated peptides were detected by both methods, whereas three additional, unique phosphopeptides were detected with SELDI-MS, and two with nano-LC-ESI-MS. When SELDI-MS/MS was used 6 individual phosphoserine residues were detected (theoretically, at least 20 phosphorylation sites among the 4 casein variants are possible), and 5 individual sites were detected with LC-MS/MS. Nanoparticles of ZrO2 were employed for phosphopeptide enrichment because of their high surface area-to-volume ratio (Zhou et al., 2007). After enrichment with these nanoparticles, the abundance of phosphopeptides was two orders of magnitude greater than the abundance of nonphosphopeptides, and this method can be applied to femtomole levels of protein digests (5 500 fmol of b-casein digest). Application of this technique to the analysis of a tryptic digest of mouse liver lysate with nano-LCMS/MS enabled identication of 248 phosphorylation sites from 140 phosphopeptides.
monophosphopeptide peak (m/z 2062). The authors also showed that 2,5-DHB in 1% phosphoric acid improved the detection of the tetraphosphorylated peptide from the membrane relative to the use of an a-cyano-4-hydroxycinnamic acid matrix (a-CHCA); those results are consistent with a prior report (Wang et al., 2007). Most likely, multiply phosphorylated peptides bind more strongly to the alumina membrane than monophosphorylated peptides, and 2,5-DHB and phosphoric acid are needed to elute these multiply phosphorylated species into the matrix. In conventional MALDI, Kjellstrom and Jensen noted that the addition of phosphoric acid to 2,5-DHB matrix solutions can enhance phosphopeptide signals, including those due to tetraphosphopeptides (Kjellstrom & Jensen, 2004). A very recent report demonstrates the potential of Nb2O5 as an enrichment medium (Ficarro et al., 2008). With simple samples containing 1 pmol of standard phosphopeptides, recoveries of phosphopeptides ranged from 50% to 100%. The use of lactic acid or 2,5-DHB in loading buffers decreased the binding of nonphosphopeptides to the resin, and enrichment of phosphopeptides from a cell lysate revealed several hundred putative phosphorylation sites. Moreover, approximately 30% of these phosphorylation sites were not found with enrichment on TiO2, and approximately 30% of the phosphorylation sites found with TiO2 enrichment were not identied with Nb2O5. Specic amino acids were associated with sequences of peptides found on each of the metal oxides. Thus, the combination of enrichment on TiO2 and Nb2O5 allows for identication of far more phosphopeptides than the use of either enrichment material separately.
3. Enrichment with Aluminum Hydroxide, Alumina, and Niobium (V) Oxide

Compared to studies of TiO2 and ZrO2, relatively little work has focused on phosphopeptide enrichment with alumina and Al(OH)3. However, enrichment with Al(OH)3 allowed detection of eight phosphorylated peptides from an a-casein digest, whereas nonphosphopeptide signals were greatly reduced compared to conventional MALDI-MS analysis (Wolschin, Wienkoop, & Weckwerth, 2005). More recently, porous anodic alumina membranes were utilized as enrichment materials for phosphopeptides (Wang et al., 2007). Such membranes were previously used to desalt biological samples (Wang, Xia, & Guo, 2005). Pieces of the membrane were incubated in a peptide mixture for 6 hr and subsequently rinsed with water and acetonitrile for 20 min. After adding the matrix, the membrane was attached to a stainless steel MALDI plate for MS analysis. Although these membranes showed high specicity for monophosphorylated (FQpSEEQQQTEDELQDK, m/z 2062, and FQpSEEQQQTEDELQDKIHPF, m/z 2556) and tetraphosphorylated (RELEELNVPGEIVEpSLpSpSpSEESITR, m/z 3122) peptides from a b-casein digest, the amount of digest required to detect all three peptides was 20 pmol. (The peak at m/z 2556 was likely due to chymotrypsin cleavage.) A b-casein digest that contained 400 fmol was also analyzed, but only produced a single
IV. NEW PLATFORMS AIMED TO SIMPLIFY THE ENRICHMENT PROCEDURE

The previous section provided many examples of the potential of IMAC and MOAC for phosphopeptide enrichment. However, such procedures frequently require the use of chromatography columns and multiple sample-handling steps that complicate the analysis and present the opportunity for sample losses on the column walls. In some cases, chromatography might not be appropriate for small sample volumes. One possibility to process small samples is the use of pipette tips lled with selective resin, as discussed in the MOAC section above. The subsections below present two alternative platforms, magnetic beads and MALDI plates coated with afnity materials.
A. Magnetic Beads
The use of magnetic beads for phosphopeptide enrichment is attractive because the beads can be easily collected using an external magnetic eld. The development of nano-sized magnetic beads makes the technique even more attractive because the high surface area-to-volume ratio provides a high binding capacity. Thus far, beads have been modied with both metal afnity complexes and metal oxides. Much of this work was reviewed very recently (Han, Ye, & Zou, 2008), so we only present a few highlights here.
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1. Metal Afnity Beads (Including Zirconium Phosphonate)

In 2007, Fe(III) and Ga(III) IMAC magnetic beads were prepared using a tetradentate ligand (not specied), and enrichment with these modied beads was compared with enrichment by other materials, including magnetic beads derivatized with IDA and metal oxides (Liang et al., 2007). The isobaric tag for relative and absolute protein quantitation (iTRAQ) labeling technique was used along with MALDI-MS/MS to determine the recovery of simple mixture of phosphopeptides. A recent review describes iTRAQ applications towards phosphoproteomics (Smith & Figeys, 2008). The new Fe(III) IMAC magnetic beads outperformed both similar magnetic beads charged with Ga(III) and a commercial ZrO2 resin (Calbiochem, Gibbstown, NJ), both of which gave phosphopeptide recoveries that were only 10% of that obtained with the Fe(III) IMAC material. Recoveries achieved with Fe(III) IMAC magnetic beads were almost identical to those of TiO2 materials (Liang et al., 2007), including TiO2 spheres from GL Sciences that have been used in other studies (Larsen et al., 2005; Thingholm et al., 2006). Additionally, MALDI-MS signals due to nonphosphorylated peptides were 5070% less when enrichment was performed with the new Fe(III) IMAC magnetic beads rather than a pipette tip that contained a commercial IMAC resin with Fe(III)IDA complexes. Detection limits were ca. 5 fmol. Immobilized metal afnity chromatography (IMAC) and TiO2-coated magnetic beads were also compared by enriching phosphopeptides from an in-gel digest of tyrosine-immunoprecipitated (IP) phosphoprotein from cell lysates (Liang et al., 2007). Proteins that contain phosphotyrosine residues were targeted in this study because they represent <2% of the phosphoamino acids in a cell (Hubbard & Cohen, 1993; Olsen et al., 2006; Zhang & Neubert, 2006). Signals due to phosphopeptides were dominant over signals of nonphosphorylated peptides, and the MALDI mass spectra looked similar for enrichment by either the new IMAC or TiO2-coated beads. Additionally, this study identied six new phosphorylation sites upon epidermal growth factor stimulation. Fe(III)IDA-coated magnetic beads were also synthesized with particle diameters of only 15 nm to increase the surface areato-volume ratio $600-fold relative to micron-sized particles (Tan et al., 2008). In MALDI-MS analyses of 2 pmol, 1 pmol, 100 fmol, and 20 fmol of a-casein digest, the Fe(III)IDA magnetic nanoparticles enabled detection of 17, 13, 7, and 2 phosphopeptides, respectively. In contrast, at one pmol of a-casein digest, the use of the commercial IMAC resin and MALDI-MS detected 5 phosphopeptides, whereas at 100 fmol, no phosphopeptides were detected. Additionally, when 2 pmol of a-casein digest was analyzed in the presence of 20 pmol of BSA digest, the magnetic beads facilitated detection of 14 phosphopeptides, whereas the commercial IMAC resin revealed half that number. The enrichment protocols used for the two materials were essentially identical; however, the loss of commercial resin during the enrichment steps (rinsing and elution) might have occurred because no mechanism was used to securely retain the particles while the supernatant was drawn off from the bead slurry. The lowest amount of the synthetic monophosphopeptide that could be analyzed with the magnetic Fe(III)IDA nano-
particles and MALDI-MS was 5 fmol (Tan et al., 2008). After a digest of plasma membrane proteins from mouse liver was fractionated with SCX chromatography and desalted with a C18 cartridge, the samples were analyzed with the IMAC magnetic nanoparticles and nano-LC-MS/MS. This analysis resulted in the identication of 217 phosphorylation sites from 158 phosphoproteins. The synthesis and use of zirconium phosphonate-modied substrates (monolithic capillary columns, polymer beads, MALDI plates, and magnetic beads) as new IMAC materials for phosphopeptide enrichment and subsequent MS analysis have been reported in the last few years (Zhou et al., 2006; Dong et al., 2007; Feng et al., 2007; Wei et al., 2008). The most recent of these publications involved the use of magnetic nanoparticles ($70-nm particle diameter) modied with Zr(IV)-phosphonate complexes to enrich femtomole levels (50500 fmol) of b-casein phosphopeptides (Wei et al., 2008). The detection limit with the Zr(IV)-phosphonate magnetic beads and MALDI-TOF-MS was $50 fmol based on the detection of a single monophosphorylated peptide (m/z 2062) from a b-casein digest. However, in the presence of a 100-fold excess of BSA digest, signals due to a number of nonphosphorylated peptides were observed in the mass spectrum when phosphopeptides were enriched from 500 fmol of b-casein digest. With iTRAQ reagents, the capacity of the nanoparticles and the phosphopeptide recovery of a standard protein, FLpTEYVATR, were determined to be 141 pmol/mg and $53%, respectively. Lastly, to show the applicability of this enrichment method towards large-scale biological samples, a SCX chromatographic fraction from digested proteins from a Chang liver cell extract was enriched with the Zr(IV)-phosphonate magnetic nanoparticles and analyzed using nano-LC-ESI-MS. This method detected 22 phosphorylated peptides from one SCX fraction (buffer contained 1 M NaCl). Enrichment with commercial PHOS-select Fe(III)-IMAC beads from Sigma allowed detection of only six phosphopeptides.
2. Metal Oxide-Coated Beads

The use of magnetic nanoparticles coated with metal oxides (e.g., titania, zirconia, alumina, and gallium oxide) to isolate phosphopeptides began in 2005 (Chen & Chen, 2005). As shown in Figure 9, magnetic (Fe3O4) nanoparticles were coated with a thin layer of SiO2 via either TEOS or sodium silicate, and the metal oxide, TiO2, ZrO2, Al2O3, or Ga2O3, was then formed on the silica with titanium butoxide, zirconium butoxide, aluminum isopropoxide, or gallium isopropoxide, respectively (Chen & Chen, 2005, 2008; Chen et al., 2007; Lo et al., 2007; Li et al., 2008). This procedure typically formed $50-, $170-, and $20nm diameter titania-, zirconia-, and alumina-coated Fe3O4 particles, respectively. Similar ZrO2- and Al2O3-coated magnetic nanoparticles with diameters of $280 nm have also been prepared (Li et al., 2007a,b). The protocol to use the metal oxide-coated magnetic nanoparticles to enrich phosphopeptides is fairly simple. For example, 24 mg of magnetic beads was mixed with $50 mL of protein digest in 0.15% TFA in a microcentrifuge tube, incubated for 30 sec, rinsed with 50% acetonitrile that contained 0.15% TFA, and the solution was decanted while using a magnet
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FIGURE 9. Fabrication of TiO2-coated magnetic nanoparticles (procedure from Chen & Chen, 2005,
2008). Zirconium, aluminum, and gallium oxide-coated magnetic nanoparticles were prepared in a similar fashion (Chen et al., 2007; Li et al., 2007a,b, 2008; Lo et al., 2007; Chen & Chen, 2008).
to secure the nanoparticles to the wall of the tube (Chen et al., 2007). The beads were either applied to a MALDI plate without the addition of matrix (Fe3O4/TiO2 surface-assisted laser desorption/ionization, SALDI) or they were mixed with 2,5DHB that contained phosphoric acid and applied to the MALDI plate for direct MALDI-TOF-MS analysis (Chen & Chen, 2005). The Fe3O4/TiO2 SALDI method is attractive because no elution step or matrix is necessary. The estimated binding capacity of alumina-coated magnetic nanoparticles is 60 mg of phosphopeptide per milligram of nanoparticle (Chen et al., 2007). Using this capacity, 25 mg of magnetic beads could isolate 1.5 mg of phosphopeptide, which for a phosphopeptide with a molecular weight of 2500 Da, corresponds to 600 pmol. This high capacity could lead to nonspecic binding. The binding capacities for the other metal oxide-coated magnetic nanoparticles were not specied. Most studies of magnetic beads employed relatively large amounts of simple phosphopeptide mixtures such as a- and bcasein digests. Recently, magnetic nanoparticles coated with TiO2 and Al2O3 were used to enrich phosphopeptides from three batches of diluted human serum with only a 30-sec incubation time (Chen & Chen, 2008). Although the different specicities of the two metal-oxide coated magnetic materials were apparent from variations in the relative peak intensities in the mass spectra, both particles enriched four phosphorylated variations of brinopeptide A (DpSGEGDFLAEGGGV at m/z 1390, ADpSGEGDFLAEGGGV at m/z 1460, DpSGEGDFLAEGGGVR at m/z 1546, and ADpSGEGDFLAEGGGVR at m/z 1617). Additionally, a peak at m/z 2753 was present in the analysis of the three batches of blood serum only with the TiO2-coated magnetic nanoparticles. This peak was due to a nonphosphorylated human serum albumin peptide (DAHKSEVAHRFKDLGEENFKALVL). However, because no 2,5-DHB was used in the loading or rinsing solutions, the nonspecic adsorption observed with the TiO2 magnetic nanoparticles might be reduced if the appropriate excluder were included in these solutions.
for minimal sample handling, high throughput, and lower sample loss than conventional resin-based techniques. With a conventional IMAC technique, 1015% of phosphopeptides were lost during the rinsing step, an additional 1020% were still retained on the IMAC column after elution, and another 1020% of the phosphopeptides were lost when samples were desalted with C18 material (Kokubu et al., 2005). The on-plate enrichment process should result in less sample loss because it simply consists of incubation on the plate, rinsing of the sample on the plate, and addition of matrix. The initial modication of MALDI sample supports as afnity devices for the isolation of biological molecules with subsequent analysis by MS began in the early 1990s and was carried out by a number of groups (Hutchens & Yip, 1993; Papac, Hoyes, & Tomer, 1994; Brockman & Orlando, 1995, 1996; Dogruel, Williams, & Nelson, 1995; Nelson et al., 1995). Subsections below describe isolation of phosphopeptides on MALDI plates modied with both IMAC functionalities and metal oxides.
1. IMAC MALDI Plates

a. Commercial MALDI afnity chips Ciphergen Biosystems (Palo Alto, CA) developed a ProteinChip System that consists of a ProteinChip Reader (i.e., MALDI linear TOF mass spectrometer), ProteinChip Software, and related accessories that are used in conjunction with ProteinChip Arrays (Tang, Tornatore, & Weinberger, 2004). A number of reports described the use of these chips for phosphorylated peptide and protein purication (Cardone et al., 1998; Davies, 2000; Laine et al., 2000; Roig et al., 2000; Mortier et al., 2001; Stoica et al., 2001; Tassi et al., 2001; Espina et al., 2004; Liu et al., 2004; Thulasiraman et al., 2004; Bowley et al., 2005; Ge, Gibbs, & Masse, 2005; Head et al., 2005; Righetti et al., 2005; Wu, Jin, & Marsh, 2005; Le Bihan et al., 2006; Bodega et al., 2007; Akashi & Yamori, 2007; Akashi et al., 2007). One example was the use of an IMAC-Gallium ProteinChip Array (IMAC chips were charged with a solution of 50 mM gallium nitrate) for the simultaneous analysis of a mixture of three synthetic monophosphorylated peptides, KRPpSQRHGSKY, TRDIYETDYpYRK, and KRELVEPLpTPSGEAPNQALLR, and their nonphosphorylated counterparts (Thulasiraman et al., 2004). The nonphosphorylated peptides (13 pmol) were in a 10-fold excess compared to the phosphorylated analogs, but all three phosphorylated peptides
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B. On-Plate Enrichment for MALDI-MS

This section presents recent developments in the on-plate afnity capture of phosphopeptides for subsequent MALDI-MS analysis. On-plate enrichment is advantageous because it allows
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showed greater signals in the mass spectrum than their nonphosphorylated counterparts. Additionally, peptide substrates phosphorylated with three specic kinases could be detected simultaneously with the IMAC ProteinChip Array. The use of these chips requires the Ciphergen SELDI ion source, which can be coupled to other commercial mass spectrometers. These chips and equipment are currently available through BioRad (Hercules, CA). Qiagen (Valencia, CA) offers phosphopeptide-afnity plates that are compatible with several MALDI mass spectrometers. The sample wells of the Qiagen IMAC Mass Spec Focus Chips contain a hydrophobic zone that encloses up to 35 mL of sample within the well along with an afnity zone that contains ligands that, when charged with a solution of Fe(III), capture the phosphopeptides. When the bound phosphopeptides are eluted from the afnity zone with matrix, they are concentrated or focused into a 0.6-mm diameter analysis zone, which is more hydrophilic than the afnity zone. Although the afnity ligands on the chip are not described, in 2006 Qiagen presented a poster at the American Society for Mass Spectrometry meeting on the use of chips modied with NTA self-assembled monolayers (SAMs) for phosphopeptide purication and concentration (Belisle et al., 2006). We recently applied these Qiagen IMAC chips towards the recovery of 125 fmol of a synthetic phosphopeptide from a 1 pmol protein-digest mixture (BSA, phosphorylase b, and esterase) (Dunn et al., 2008). Using an isotopic internal standard, the recovery of the phosphopeptide from a protein digest mixture was 13%, and minimal signals due to nonphosphorylated peptides were observed. More recent results from a digest containing threefold higher reagent concentrations showed a recovery of 30%. We estimate that the binding capacity of the Qiagen IMAC chip is ca. 5 pmol. This estimate assumes an afnity zone area of 7 mm2 and binding of a monolayer of phosphopeptide to this zone with a density of 1 peptide per 2.5 nm2. Although the Qiagen IMAC chip has excess binding capacity for a 125 fmol sample, recovery was still 30% or less.
b. Monolayers of metal-ion complexes immobilized on MALDI surfaces A number of research groups have prepared custom MALDI plates with IMAC functionalities. The immobilization of SAMs derivatized with NTA analogs was rst reported in 1996, and a number of subsequent studies demonstrated the fabrication of similar monolayer lms (Sigal et al., 1996; Stora et al., 1997; Scheibler et al., 1998; Kada et al., 2002; Luk et al., 2003; Makower et al., 2003; Rigler et al., 2003; Wegner et al., 2003; Gamsjaeger et al., 2004; Lee et al., 2004; Rigler, Ulrich, & Vogel, 2004; Trammell et al., 2004; Maly et al., 2004a,b; Johnson & Martin, 2005; Tinazli et al., 2005; Klenkar et al., 2006; Valiokas et al., 2006; Ataka & Heberle, 2006; Ataka, Richter, & Heberle, 2006). These NTA SAMs have been employed in the study of histidinenickel, proteinprotein, proteinantibody, protein DNA, or proteinligand interactions, and for biotechnology applications including microarrays, biosensors, catalysis, and biocompatible coatings. However, it was not until recently that NTA SAMs immobilized on gold MALDI plates were used to capture phosphorylated peptides for direct MALDI-TOF-MS analysis (Shen et al., 2005). In short, a SAM of 1,8-octanedithiol was deposited on a gold substrate, and allowed to react with maleimide-terminated NTA (N-[5-(30 -maleimidopropylamido)1-carboxypentyl]-iminodiacetic acid) as shown in Figure 10. The immobilized NTA ligand was subsequently charged with Ga(III) from 200 mM gallium nitrate. Shen and co-workers used these Ga(III)NTASAM-modied plates for on-probe enrichment of a mixture that contained two synthetic phosphopeptides, DLDVPIPGRFDRRVpSVAAE and KIGDFGMTRDIYETDpYpYRKGGK, and four nonphosphorylated peptides, angiotensin I, ACTH 117, ACTH 1839, and ACTH 739 (ACTH is an abbreviation for adrenocorticotropic hormone). In the conventional MALDI-MS analysis of the mixture, the four nonphosphorylated peptides gave stronger signals than the monophosphorylated peptide, and the diphosphorylated peptide was not detected. In contrast, both phosphopeptides were detected when the same mixture was analyzed after capture on the Ga(III)NTASAM-modied probe, whereas the peaks
FIGURE 10. Formation of a self-assembled monolayer of octanedithiol and derivatization of this
monolayer by reaction with maleimide-terminated NTA (procedure from Shen et al., 2005). Further NTA complexation of Ga(III) or Fe(III) is not shown here.
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due to the nonphosphorylated peptides were eliminated or reduced. (The matrix employed in their analyses was a-CHCA.) However, even though the MS signal due to the nonphosphorylated peptide ACTH 1839 was signicantly reduced, it was still more intense than the peaks due to the phosphorylated peptides. A tryptic digest of b-casein was also analyzed with the Ga(III)NTASAM-modied plates (Shen et al., 2005). Conventional MALDI-MS analysis generated signals for a few nonphosphorylated peptides in addition to a relatively lowintensity peak for the monophosphorylated peptide. The signal for the monophosphorylated peptide increased approximately threefold when the SAM-modied probe was used rather conventional MALDI-MS analysis. However, no signal for the tetraphosphorylated peptide was observed in either the conventional analysis or analysis after enrichment on the modied plate. Nonspecic adsorption of nonphosphorylated peptides was also apparent in the mass spectrum when the digest was applied to the Ga(III)NTASAM-modied probe. The authors stated that they saw better reproducibility in the mass spectra when Ga(III) rather than Fe(III) was used as the metal ion. In a completely different synthetic method, Xu and coworkers derivatized a porous silicon surface with a monolayer of IDA-1,2-epoxy-9-decene via a photochemical reaction (Xu et al., 2006). In brief, excess IDA was allowed to react with 1,2-epoxy9-decene to form a photochemically reactive IDA derivative. The electrochemically etched porous silicon substrate was immersed in a solution of the IDA derivative, and was exposed to light from a 1000-W Hg lamp for 2 hr, after which IDA-1,2-epoxy-9-decene derivative was assumed to be immobilized onto the silicon surface as shown in Figure 11. The IDA-derivatized silicon surface was immersed in a 100 mM FeCl3 solution to form the Fe(III)IDA complex. The Fe(III)IDA-derivatized porous silicon plates were used to analyze tryptic digests of b-casein with a matrix solution that contained 2,5-DHB and 1% phosphoric acid. The conventional mass spectrum of the digest revealed the presence of the three phosphorylated peptides with m/z 2062, 2556, and 3122 along with several peaks due to nonphosphorylated peptides. When a one pmol digest was analyzed after capture on the Fe(III)IDA-modied silicon probe, only peaks due to the phosphorylated peptides were present. However, there was signicant background noise in the mass spectrum, and the signal intensity due to the phosphopeptides decreased compared to the conventional mass spectrum. When the amount of digest was decreased from 1 pmol to 300 fmol, all three phosphopeptides were still detected when capture on the modied silicon plate was used, and nonspecic adsorption from other peptides was minimal. At the lower amount of digest, the peak intensities
of the phosphopeptides were higher than those observed in the conventional MALDI mass spectrum of the same amount of digest. c. Polymer-modied MALDI plates To increase the binding capacity for metal-afnity-based on-plate enrichment, we recently modied Au plates with polymer lms that contain metal-ion complexes. Initially, we used patterned plates with small (0.2-mm diameter) spots of a hydrophilic polymer, poly(acrylic acid) (PAA), surrounded by a hexadecanethiol monolayer (Xu, Bruening, & Watson, 2004). Immersion of the plate in an Fe(NO3)3 solution created the Fe(III)PAA complex. These patterned metal-afnity probes served to both purify and concentrate the analyte as shown in Figure 12 (Xu, Watson, & Bruening, 2003; Xu, Bruening, & Watson, 2004). When the patterned, Fe(III)PAA-modied plate was used to enrich the phosphopeptides from one pmol of ovalbumin digest, signals due to the phosphopeptides were enhanced compared to those observed in the conventional MS analysis; however the mass spectrum of the enriched sample still contained large signals due to nonphosphorylated peptides (Xu, Bruening, & Watson, 2004). The matrix used in these phosphopeptide analyses was a-CHCA. The surface might not be completely saturated with Fe(III), and excess negatively charged carboxylate groups might bind positively charged, nonphosphorylated peptides via electrostatic interactions. Perhaps more stringent rinsing, including acetic acid and/or acetonitrile, could have alleviated nonspecic adsorption. Poly(acrylic acid) (PAA)-modied plates derivatized with poly(ethyleneimine) (PEI) were also used to capture phosphorylated peptides from 100 fmol of b-casein digest (Xu, Bruening, & Watson, 2004). The signals due to the monophosphorylated and tetraphosphorylated peptides of b-casein dominated the mass spectrum of a digest captured on the PEI-modied plate. In this case, enrichment likely occurred because of interactions between positively charged PEI and the negatively charged phosphopeptides. In addition to the phosphate groups, there are several acidic amino acid residues (D and E) in the tryptic phosphopeptides of b-casein, and these groups could also be attracted to the positively charged PEI surface. It is doubtful that an electrostatic technique could be used to purify typical phosphopeptides with fewer acidic sites in moderately complex mixtures. To enhance the selectivity of polymer-modied plates, we derivatized thin PAA lms ($30 A) with the Fe(III)NTA complexes that are frequently employed in IMAC (Dunn, Watson, & Bruening, 2006). Deposition of protein digests on the polymer-modied plates, followed by a rinse with an acetic acid solution, addition of matrix, and subsequent analysis with MALDI-MS produced mass spectra that were dominated
FIGURE 11. Immobilization of a monolayer of an IDA derivative on porous silicon using photochemistry
(procedure from Xu et al., 2006).
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FIGURE 12. Fabrication of polymer-modied gold MALDI plates for phosphopeptide capture and
concentration (Xu, Watson, & Bruening, 2003). A hydrophobic pattern is created rst, followed by the immobilization of polymers onto the 0.2-mm diameter gold wells. HDT hexadecanethiol, PDMS popoly(dimethylsiloxane). Reproduced from Xu, Watson, & Bruening, 2003. Copyright 2003, American Chemical Society, used by permission.
by peaks due to singly and multiply phosphorylated peptides from b-casein and ovalbumin digests. We have also examined on-plate enrichment with poly (2-hydroxyethyl methacrylate) (PHEMA) brushes modied with Fe(III)NTA complexes (Dunn et al., 2008). These brushes are 10-fold thicker than the Fe(III)NTAPAA lms mentioned above, and thus have greater binding capacities. Ellipsometric studies of phosphoangiotensin binding to these lms revealed a binding capacity of $0.6 mg/cm2, and enrichment with the Fe(III)PHEMANTA brushes allowed MALDI-MS detection down to 15 fmol of phosphopeptide (based on the detection of a monophosphopeptide (m/z 2062) from a b-casein digest). The high sensitivity is presumably due to a relatively high binding capacity. Using an isotopically labeled internal standard added to the MALDI plates after rinsing and prior to addition of matrix, we determined a recovery of $70% for a synthetic monophosphopeptide with the Fe(III)NTAPHEMA-modied plates, even when the digest contained one pmol of BSA, phosphorylase b, and esterase. Unfortunately, recovery does decrease greatly in the presence of higher amounts of digest reagents. In contrast, thin lms of Fe(III)NTAPAA recovered just $23% of the synthetic phosphopeptide, and a monolayer of Fe(III)NTA recovered only $9%. We also compared the performance of the Fe(III)NTAPHEMA-modied plates with commercially available IMAC and metal oxide materials. The phosphopeptide recoveries of the commercial enrichment methods were typically threefold lower than that of the PHEMA brushes, with the exception of a TiO2 microtip, which had a similar recovery of $70%. However, the TiO2 microtip exhibited signicant impurities in its eluent. Lastly, the analysis of a synthetic
44
diphosphorylated peptide resulted in $100% recovery from a protein digest mixture that contained BSA, phosphorylase b, and esterase when enrichment was performed using the Fe(III) NTAPHEMA-modied plates. Because they have multiple binding sites, diphosphorylated peptides evidently have a higher afnity for the Fe(III)NTA complex than do monophosphorylated peptides. Recently, a plastic MALDI chip that selectively captures phosphorylated species was fabricated (Ibanez, Muck, & Svatos, 2007). The chips were prepared by copolymerizing methyl methacrylate and acrylic acid N-hydroxysuccinimide ester, which can react with amino-terminated NTA to provide the immobilized ligand (Ibanez, Muck, & Svatos, 2007). Any remaining active ester sites reacted with tris(hydroxymethyl)aminomethane, and the NTA was charged with Ga(III) or Ni(II) to analyze phosphorylated peptides or proteins, respectively. Chips charged with Ga(III) were selective for phosphopeptides from an a-casein digest, whereas Ni(II) was better for phosphoprotein recovery based on the enrichment of ve pmol of a-casein from an equimolar mixture that contained myoglobin and carbonic anhydrase I (phosphoprotein adsorption occurred at pH $8). The Ga(III)NTA plastic MALDI chips were used to analyze one pmol of phosphorylated angiotensin from a nonphosphorylated BSA digest (3.5 pmol). After the sample was applied to the modied plate, rinsed to remove unbound species, and mixed with matrix, the dominant signal in the mass spectrum was that due to phosphoangiotensin, whereas many signals due to BSA were reduced compared to conventional MALDI-MS. This technique was also used to analyze $2 pmol of an a-casein digest. Although the plate was
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able to retain eight phosphopeptides, some of the strongest peaks in the mass spectrum were due to nonphosphorylated peptides. Thus, the polymer plate appears to be subject to nonspecic adsorption.
the BSA digest (10- and 100-pmol amounts). Nonspecic adsorption is a frequent limitation of IMAC, but the extent of nonspecic adsorption likely depends on the rinsing and loading protocol.
d. Zirconium phosphonate lms A number of studies demonstrated the formation of zirconium phosphonate lms on various surfaces because of potential applications in catalysis, sensing, electronics, protein immobilization, and separations (Lee et al., 1988; Putvinski et al., 1990; Hong, Sackett, & Mallouk, 1991; Byrd, Pike, & Talham, 1993; Frey, Hanken, & Corn, 1993; Byrd et al., 1994; Nixon et al., 1999; Cleareld et al., 2000; Kohli & Blanchard, 2000; Kumar & Chaudhari, 2000; Benitez et al., 2002; Nonglaton et al., 2004; Mazur, Krysinski, & Blanchard, 2005). Zr(IV) binds strongly to phosphonate monolayers due to metal ion-ligand crosslinking (Zr(IV) ions coordinate to more than one phosphonate molecule), (Byrd, Pike, & Talham, 1993; Byrd et al., 1994) and multilayer Zr(IV) phosphonate lms can also be prepared (Nixon et al., 1999; Cleareld et al., 2000; Kohli & Blanchard, 2000). With regard to phosphopeptide enrichment, Zhou and coworkers used zirconium phosphonate monolayers immobilized on porous silicon as phosphopeptide afnity probes for MALDITOF-MS (Zhou et al., 2006). To form the phosphonate-silicon surface, an etched silicon substrate was rst placed in a solution of phosphorous oxychloride (POCl3) and 2,4,6-trimethylpyridine (collidine). Subsequently, the surface was charged with Zr(IV) by immersing the phosphonate-modied silicon substrate into 20 mM ZrOCl2, and these modied plates were used to analyze digests of b-casein with 2,5-DHB in 1% phosphoric acid as a matrix. Enrichment of phosphopeptides from the b-casein digest with the Zr(IV)-phosphonate silicon substrate yielded signals for mono (m/z 2062, 2556) and tetraphosphorylated (m/z 3122) phosphopeptides at the 2-pmol and 20-fmol levels. When 2 fmol of b-casein was analyzed using the porous silicon plates, only phosphopeptide peaks at m/z 2061 and 2556 were present. Signals due to nonphosphorylated peptides were minimal at all digest concentrations. Fifteen phosphorylated peptides were isolated from a 2pmol a-casein digest with the Zr(IV)-phosphonate-modied silicon plate (Zhou et al., 2006). A number of these peptides arise from missed cleavages. Even though 14 of the phosphorylated peptides were observed in the conventional MALDI mass spectrum, the modied silicon plate simplied the mass spectrum by eliminating nearly all signals due to nonphosphorylated peptides. Additionally, b-casein was combined with a tryptic digest of BSA, a nonphosphorylated protein, and analyzed at b-casein to BSA molar ratios of 1:1, 1:10, and 1:100 (the amount of b-casein in the digest was maintained at 1 pmol). Impressively, the use of the derivatized porous silicon plates eliminated nearly all signals that corresponded to peptides from BSA. However, when the amount of BSAwas 100-fold greater than b-casein, the signals for the monophosphorylated peptides (m/z 2061 and 2556) dramatically decreased. For comparison, they also applied the mixture to Fe(III)-IMAC beads (Poros MC beads, Applied Biosystems, Foster City, CA) that contain an IDA ligand. The Fe(III)IDA beads suffered from nonspecic adsorption at high amounts of
2. MALDI Plates Modied with Metal Oxides

a. Zirconium oxide-functionalized MALDI surfaces Recently, zirconium oxide was immobilized on a stainless steel plate via collisions of Zr(IV)-n-propoxide cations from an ESI source onto an oxidized surface (Blacken et al., 2007). This procedure resulted in zirconium oxide spots with a diameter of 5.5 mm, and these spots showed afnity toward phosphopeptides in a 1 pmol a-casein digest. The digest was applied to the modied surface, incubated for 20 min, rinsed with an aqueous solution of 0.1% TFA in 20% acetonitrile, and dried. Addition of matrix and MALDI-MS resulted in a mass spectrum with greatly reduced signals (>25-fold reduction compared to conventional MALDI-MS) due to nonphosphopeptides, while two monophosphorylated peptides (m/z 1660 and 1951) produced two of the most intense peaks in the mass spectrum. However, the overall signal intensity of these two phosphopeptides decreased by a factor of two compared to peaks in the conventional analysis of the digest. Although this functionalized surface retained phosphorylated peptides, several synthetic peptides that contained methionine or tryptophan residues were readily oxidized, perhaps because of oxidation catalysis by zirconia. The authors suggested the addition of a reducing agent such as dithiothreitol to the loading solution to minimize any amino acid oxidation. b. Immobilized titanium dioxide on MALDI plates As described in Section II.C.1., TiO2 is very effective in phosphopeptide enrichment. In an effort to simply phosphopeptide isolation, TiO2-coated gold nanoparticles immobilized on a glass slide were employed for on-plate enrichment and analysis of phosphopeptides with MALDI-TOF-MS (Lin, Chen, & Chen, 2006). The scheme in Figure 13 shows the fabrication of the TiO2-gold-nanoparticle (TiO2-gold-NP) glass plate. Using the Frens method (Frens, 1973), gold nanoparticles were prepared with an average diameter of 26 nm. To prepare the glass slide for the attachment of the gold nanoparticles, a thin lm of TMSPED (N-[3-(trimethoxysilyl)propyl]ethylenediamine) was bound to the oxidized glass surface. Gold nanoparticles were immobilized onto the TMSPED-treated surface, and a solution of titanium isopropoxide was spin-coated onto the substrate, followed by annealing. The coverage of the 26-nm diameter gold nanoparticles on the glass slide was estimated to be $620 nanoparticles/mm2 (34% coverage) (Lin, Chen, & Chen, 2006). Tryptic digests were applied to the modied glass plates in 500mL aliquots, and after the sample had incubated for 1 hr, the surface was rinsed to remove any unwanted species prior to the addition of 2,5-DHB that contained H3PO4 and MALDITOF-MS. The TiO2-gold-NP-modied glass plates were used to analyze b-casein digests that contained relatively high amounts of protein (50 and 500 pmol) (Lin, Chen, & Chen, 2006). Three phosphorylated peptides (m/z 3122, 2556, and 2062) were captured and detected with the TiO2-gold-NP plate,
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FIGURE 13. Immobilization of titania-coated gold nanoparticles (NPs) on glass substrates (procedure
from Lin, Chen, & Chen, 2006).
but nonspecic adsorption was observed with the 500-pmol sample (500 mL of 1-mM b-casein digest). Using the modied glass plate, Lin and co-workers also analyzed two more complex samples: (1) an equimolar mixture (50 pmol of each) of cytochrome c and b-casein, and (2) milk, which contains a-S1-, a-S2-, and b-casein. Interestingly, there were almost no peaks due to peptides from cytochrome c (a nonphosphorylated protein) in the mass spectrum when the equimolar digest mixture was enriched with the TiO2-gold-NP-modied glass plate. When the milk digest was applied to the modied plate, four phosphopeptides (m/z 3122, 3008, 2556, and 1952), one due to chymotrypsin cleavage and one due to miscleavage of a-S1-casein, were detected. This number of phosphopeptides detected is quite low because a-S1- and a-S2-casein are highly phosphorylated (there are over 20 phosphorylation sites between the two proteins). If all three casein proteins are digested completely (i.e., no miscleavage), then 11 phosphorylated peptides should result. Hence, only 18% of fully cleaved, phosphorylated peptides were detected in the milk digest enriched on the titania bead-modied target. Conventional MALDI-MS analyses of the digests would be useful for comparison. More recently, an array of titanium dioxide nanoparticles was formed on a commercial stainless steel MALDI plate
by spotting an array of 2-mL droplets of a 100-mg/mL TiO2 suspension onto the surface, and heating at 4008C for 1 hr (Qiao et al., 2007). The selectivity of this metal oxide afnity plate was demonstrated by enriching phosphopeptides from b-casein digests at picomole down to low femtomole levels in just 30 min (sample incubation time). At relatively high digest concentrations (850 nM), six phosphopeptides from a-casein impurities (m/z 1467, 1540, 1595, 1661, 1928, and 1952) in addition to three b-casein phosphopeptides (m/z 2062, 2556, and 3122) were enriched from a b-casein digest. For a lower incubation time of 5 min, a single monophosphorylated peptide was still detected at 30 fmol of b-casein digest. Most impressively, the three b-casein phosphopeptides at 26 nM were still enriched in the presence of a 100-fold excess of BSA digest (30-min incubation). Polymeric MALDI plates that contained channels packed with titanium dioxide were used to demonstrate the feasibility of this modied plate geometry to enrich phosphopeptides (Ekstrom et al., 2007). Similar plates for solid-phase microextraction were used previously by the same group (Ekstrom et al., 2004, 2006). Plates fabricated with pyramidal-shaped channels were packed with titania (Fig. 14) to selectively enrich and concentrate phosphopeptides from one pmol and 100 fmol b-casein digests
FIGURE 14. Cross-section of a conducting polymer MALDI plate, showing one pyramidal-shaped
channel packed with 50-mm diameter TiO2 particles (Ekstrom et al., 2007). The inlet and outlet have widths of 1 mm and 50 mm, respectively. All solutions are pulled through the channel using a vacuum.
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(Ekstrom et al., 2007). A vacuum was used to pull all solutions (digest, wash, elution, and matrix) through the packed channels. Hence, the matrix crystallized on the rear side of the modied plate (Fig. 14), which was then analyzed with MALDI-MS. When one pmol of b-casein digest was analyzed, the monophosphopeptide (m/z 2061) and tetraphosphopeptide (m/z 3122) were retained and detected, whereas essentially all signals due to nonphosphopeptides were eliminated. When the sample amount was reduced to 100 fmol, only a weak signal due to the monophosphorylated peptide was detected. TiO2-coated magnetic nanoparticles have also been afxed to the sample wells of a conventional stainless steel MALDI plate by holding a magnet to the back of the sample plate during the sample loading, incubation (10 min), rinsing, and matrix-loading steps (Tan et al., 2007). These modied plates were used to enrich phosphopeptides from a-casein, b-casein, and ovalbumin digests. The sensitivity of this method was demonstrated by the enrichment of both the monophosphorylated (m/z 2062) and tetraphosphorylated peptides (m/z 3122) from 100 fmol of b-casein digest, and the detection limit of the monophosphorylated peptide appears to be $10 fmol. The selectivity was demonstrated by the detection of 14 phosphopeptides, 6 of which were multiply phosphorylated, from a mixture of a-casein, b-casein, ovalbumin, myoglobin and BSA digests. Conventional MALDI-MS yielded signals for only ve monophosphorylated peptides. Although the intensities of many signals due to nonphosphopeptides were reduced compared to conventional analysis, a few nonphosphopeptide peaks gave more intense signals than those of the phosphorylated peptides.
enhanced in the presence of phosphoric acid (Kjellstrom & Jensen, 2004; Stensballe & Jensen, 2004). This tetraphosphorylated peptide was not detected when TFA, rather than phosphoric acid, was used in the matrix solution. The authors suggest that the phosphoric acid-enhanced detection of phosphorylated peptides is due to a salting out effect, as PO43 is known to be the best anion for precipitating proteins. Both 2,5-DHB and a-CHCA are widely used in phosphopeptide analysis, but in our hands 2,5DHB/phosphoric acid has typically yielded stronger phosphopeptide signals than a-CHCA/diammonium hydrogen citrate. (Asara and Allison introduced ammonium salts including diammonium hydrogen citrate and ammonium acetate to the MALDI matrix for enhanced detection of phosphorylated peptides using positive-ion mode MALDI-TOF-MS; Asara & Allison, 1999.) Under the conditions we examined, sweet spots were easier to nd in a 2,5-DHB/phosphoric acid matrix than in a THAP/DAHC matrix.
V. PERSPECTIVE
Immobilized metal afnity chromatography (IMAC), MOAC, and covalent methods are all capable of selectively enriching phosphopeptides, and thus far one technique has not emerged as superior to the others. Covalent techniques are sometimes timeconsuming with many steps, but several recent studies presented simplied procedures (Tao et al., 2005; Bodenmiller et al., 2007b). Still, esterication of carboxylic acid groups is usually required for these techniques, and if the esterication is less than quantitative, this will complicate mass spectra (Yu et al., 2007; Simon et al., 2008). As mentioned previously, there will also be peptide loss during lyophilization after esterication (Stewart, Thomson, & Figeys, 2001). Metal oxide afnity chromatography (MOAC) based on adsorption to TiO2 is especially attractive, but as with all techniques, loading, rinsing, and elution solutions must be carefully selected to minimize nonspecic adsorption and to maximize the detection of both monophosphorylated and multiphosphorylated species. It appears that with appropriate reagents, it might not be necessary to esterify proteins prior to their enrichment on TiO2. However, recent work by Simon et al. suggests that methyl esterication does enhance the specicity of the technique when 2,5-DHB is used to exclude some nonspecic adsorption (Simon et al., 2008). Recently developed excluders such as phthalic acid (Thingholm et al., 2006), glycolic acid (Jensen & Larsen, 2007), and ammonium glutamate (Yu et al., 2007) seem to allow purication without the drawbacks of methyl esterication. Glycolic acid, as well as some of the other new excluders, may be more compatible with LC-MS than 2,5-DHB. Immobilized metal afnity chromatography (IMAC) might not provide the selectivity available with TiO2 enrichment, but with appropriate reagents, IMAC can be selective and sensitive for monophosphorylated and tetraphosphorylated peptides. However, some buffers and reagents such as EDTA are not compatible with IMAC, so HPLC purication may be needed prior to this technique (Jensen & Larsen, 2007). MOAC appears to be more tolerant to EDTA and other reagents such as surfactants.
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3. MALDI Matrices
The selection of the MALDI matrix affects phosphopeptide signals in MALDI regardless of whether on-plate purication is employed. Typical matrix molecules include a-CHCA, 2,5DHB, and 20 ,40 ,60 -trihydroxyacetophenone (THAP). Yang and co-workers showed that the signals of phosphopeptides in b-casein and protein kinase C (PKC)-treated mouse cardiac troponin I increased 10-fold when using THAP with ammonium citrate rather than a-CHCA as a matrix (Yang et al., 2004). We also found that THAP gives higher phosphopeptide signals than a-CHCA (Dunn, Watson, & Bruening, 2006). Hart and coworkers examined the use of both 2,5-DHB and a-CHCA to elute phosphopeptides from IMAC beads and serve as a matrix. In the analysis of an a-casein digest, DHB revealed more peptides with stronger signals. They suggested that 2,5-DHB gives reduced energy transfer to the matrix to yield more intact phosphopeptides (Hart et al., 2002). Kjellstrom and Jensen demonstrated the use of phosphoric acid, after examining ve acids (acetic acid, formic acid, TFA, heptauorobutyric acid, and phosphoric acid), as a suitable matrix additive to enhance phosphopeptide signals in MALDI TOF-MS (Kjellstrom & Jensen, 2004). In both positive and negative ionization modes, when 200 fmol of a-casein was analyzed, the use of phosphoric acid in the matrix solution yielded increased signals from all four observed phosphopeptides. In analysis of 100 fmol of b-casein digest using positive-ion mode, the ionization of the tetraphosphorylated peptide was also
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One challenge to assess the utility of different techniques is the limited pool of proteins that are examined in model systems. Many studies have focused on a- and b-casein because of the low cost of these proteins. Development of a more complete series of phosphopeptides with a wider range of residue compositions would enhance our understanding of the efciency of phosphopeptide enrichment using different systems. Additionally, the high recoveries achieved in simple protein digests are most likely not relevant to more complex samples. However, evaluation of cell lysates is challenging because the phosphopeptide composition is unknown. A combination of enrichment techniques is needed to achieve the highest phosphopeptide coverage. Bodenmiller and co-workers recently compared IMAC, MOAC (TiO2 using phthalic acid as an excluder), and covalent phosphoramidate enrichment using the same digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells (Bodenmiller et al., 2007b). After enrichment, phosphopeptides were identied with LC-ESI tandem mass spectrometry. Although the study could not say what fraction of the total phosphopeptides were identied, it did allow an important comparison of the phosphopeptides identied by the different techniques. IMAC, MOAC, and covalent enrichment identied 366, 535, and 555 phosphopeptides, respectively, but the overlap of phosphopeptides identied using the different techniques was only approximately 34%. Thus, although all of the methods showed relatively high specicity for phosphopeptides, a combination of techniques is needed for the highest phosphopeptide identication. Overall, nearly 900 phosphopeptides were identied using the combination of the three techniques. Nevertheless, this may still be only a small fraction of the total phosphopeptides in the cell. Spiking of cell lysates with a series of 1020 synthetic phosphopeptides might provide a useful system for investigating the level at which phosphopeptides can be detected as well as the comprehensiveness of the methods. The choice of enrichment platform (column, pipette tip, magnetic bead, or modied MALDI plate) will likely depend on the particular application. When trying to isolate and identify as many phosphoproteins as possible in a cell lysate, chromatographic column-based methods are required. Multiple elutions from IMAC or MOAC columns or even gradient elutions can help to simplify fractions of proteins and reveal more peptides (Simon et al., 2008; Thingholm et al., 2008). However, as mentioned above, even with a combination of methods, it is not clear what fraction of phosphopeptides can be identied in a complex sample. For heterogeneous samples, extraction with magnetic beads is attractive to avoid the need for ltration prior to enrichment. In the case of small (mL), relatively pure samples (e.g., from immunoprecipitated proteins) pipette tips and modied MALDI plates are both attractive. However, the on-plate enrichment offers advantages in terms of simplicity and minimal sample loss. With detection limits at the femtomole level, modied plates are becoming more viable for such applications. However, these techniques are not likely to prove suitable for complex samples. Additionally, it is important to carefully select the capacity of the extractant (magnetic bead, plate, or column) because when the binding capacity of the material is exceeded, there will be selective extraction of specic phosphopeptides, frequently
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multiply phosphorylated species. In contrast with too high a binding capacity, nonspecic interactions might occur. Phosphopeptide enrichment using magnetic beads and modied MALDI plates is still relatively new and has not yet gained widespread use. Overall, the potential for phosphopeptide enrichment and rapid analysis has improved dramatically over the past 5 years. A combination of techniques can reveal large numbers of phosphopeptides in complex samples, but comprehensive phosphoproteomics is still not possible. For the highest protein coverage, future phosphoproteomic techniques will likely employ multiple enrichment techniques along with two-dimensional separations, but such studies are time consuming. The combination of immunoprecipitation and enrichment should prove useful for reducing the complexity of samples, but this will not be comprehensive. At present the challenge of complete phosphoproteomics is daunting at best. Comparisons of the levels of specic phosphopeptides/phosphoproteins in different cells or in response to specic treatments are much more feasible. In such cases, simple techniques such as on-plate purication can help to rapidly identify phosphorylation sites in proteins that are isolated from complex mixtures. We expect that many future studies will focus on the use of these techniques to understand phosphorylation states in important biochemical pathways.
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Jamie D. Dunn is currently a chemist of the U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Division of Pharmaceutical Analysis in St. Louis, MO. She received her Ph.D. in Chemistry (2007) and M.S. in Chemistry and Criminal Justice from Michigan State University. Her research interests include on-plate purication for MALDIMS and drug analysis using HPLC-MS. Gavin E. Reid is an Assistant Professor in the Department of Chemistry and the Department of Biochemistry and Molecular Biology at Michigan State University. He received his PhD in Chemistry at the University of Melbourne in 2000, carried out post doctoral research at Purdue University from 20002002 and was an Assistant Member of the Ludwig Institute for Cancer Research in Melbourne, Australia from 20022004. His research interests involve fundamental and applied studies toward the development of mass spectrometry strategies for targeted proteome and lipidome analysis. Merlin L. Bruening is a Professor of Chemistry at Michigan State University. Prior to joining the faculty at Michigan State in 1997, he was an NIH-sponsored postdoctoral researcher at Texas A&M University. He received his PhD in 1995 from The Weizmann Institute of Science. His research interests lie in the development of thin lms and membranes for separations, catalysis, and analysis, including sample purication prior to analysis by mass spectrometry.
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Techniques For Phosphopeptide Enrichment Prior To Mass Spec Anal

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TECHNIQUES FOR PHOSPHOPEPTIDE ENRICHMENT PRIOR TO ANALYSIS BY MASS SPECTROMETRY

Mass Spectrometry Reviews, 2010, 29, 29 54 # 2009 by Wiley Periodicals, Inc.

DUNN, REID, AND BRUENING

III. PHOSPHOPEPTIDE CAPTURE AND ELUTION A. Immobilized Metal Afnity Chromatography