Technology Profile

2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Edgar Ngele, Martin Vollmer, Patric Hrth and Cornelia Vad
Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.
Expert Rev. Proteomics 1(1), 3746 (2004)

CONTENTS
Results & discussions Conclusion Expert opinion Five-year view Key issues References Affiliations

Author for correspondence Agilent Technologies R&D & Marketing GmbH & Co. KG, Hewlett-Packard-Str. 8, Waldbronn, Germany Tel.: +49 724 360 2663 edgar_naegele@agilent.com KEYWORDS: 2D-LC, chip LC, LC, multidimensional, nano-LC/MS, offline 2D-LC, online 2D-LC, proteomics

In this review, examples of online and offline multidimensional liquid chromatography (LC) in proteomics using an 1100 LC system (Agilent Technologies) are included and their system internal workflow will be presented and discussed. Furthermore, the use of these systems for specific applications in the analysis of proteome samples will also be discussed, depending on their complexity. An outlook on the future possibilities in proteomics with regards to new instrumentation is also considered. The proteome, which can be defined as the complete protein set of a cell, derived from a certain time point and condition of a cell, microorganism, or body fluid, is now the focus of many international research groups and pharmaceutical companies. The sequenced human genome and expression levels of messenger RNA (mRNA) do not describe the current state of a living cell in sufficient detail. This is due to the broad variety of post-translational modifications (PTM), thus, scientists discovered the importance of analyzing the actual protein composition of a biological system in order to understand complex cellular processes and networks. Proteomics as a scientific discipline emerged with the purpose to identify rapidly

complex protein patterns in a comprehensive manner [13]. The methodology required to achieve this ambitious goal must also be powerful enough to detect subtle quantitative and qualitative differences of a protein profile in order to finally identify target proteins and modified variants [4,5]. Such comprehensive proteome characterization will give new insight into cellular responses for disease pathogenesis, such as carcinogenesis, for development, as well as aging, drug action and environmental damage. In order to identify proteins from a complex mixture of 5 103 to 5 104 compounds with a dynamic range of at least 105 [6], it will be crucial to develop technologies with extremely good resolving power on one hand and with extraordinary sensitivity on the other hand. It is obvious that these challenging tasks will not be achieved with a single analytical technology but through the combination of separation and detection techniques. The current method of choice for separating complex proteomic samples is still 2D polyacrylamide gel electrophoresis (PAGE), due to its high resolving power for proteins [68]. During the 2D-PAGE procedure, the protein mixture is separated by

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Ngele, Vollmer, Hrth & Vad

isoelectric focusing (IEF) in the first dimension and by sodium Reversed phase (RP) chromatography capillary electrophoresis [20,21] dodecyl sulfate (SDS)-PAGE in the second dimension. Following gel digestion of target proteins, identification is usually achieved Size-exclusion chromatography (SEC)-RP [22] by matrix-assisted laser desorption/ionization time-of-flight Strong cation exchange (SCX) chromatography-RP [23,24] (MALDI-TOF) or LC/electrospray ionization (ESI) ion trap. Despite recent progresses of 2D-PAGE in terms of reproduci- Anion exchange chromatography-RP [25] bility, automation and quantization [911], the technique still suf Affinity chromatography (AC)-RP [26,27] fers from some major drawbacks. This holds true especially for While the latter approach is mainly applied to more specific low-abundance proteins, membrane proteins, proteins with extreme pI values and very large or very small proteins. The functional approaches such as phosphopetide analysis [28], SCXapplication of 2D-LC for such critical protein classes has been RP (e.g., with the multidimensional protein identification techdemonstrated and reviewed in a variety of reports [1214]. In addi- nology [MudPIT]) in contrast, has been successfully applied to tion, it has been applied to membrane proteins, which are often the analysis of comprehensive protein expression profiles from the targets for drug action and are therefore most promising for yeast Saccharomyces cervisiae or Plasmodium falciparum [2931]. Usually, online 2D-LC-tandem MS (MS/MS) is performed the development of novel drugs or diagnostic tests [15]. In order to overcome these major obstacles in 2D-PAGE, using a SCX column in series with a RP column. In the course LC-based separation techniques directly coupled to mass spec- of analysis tryptic peptides are eluted stepwise by injecting salt trometry (MS) detection were developed with the goal of plugs of increasing ionic strength from the SCX column in the obtaining a comparable resolution as in 2D-PAGE. The first dimension. In the second dimension these peptides are orthogonal combination of different high-performance (HP)- first trapped on a RP enrichment column and finally separated LC techniques (2D-LC or multidimensional LC), either for on an analytical RP column [24]. It has been shown that this intact proteins or for peptide mixtures, has been described in a methodology, especially at nanoscale [32], is capable of resolving variety of reports [16]. In general, peak capacity for a multidimensional separa- A Orthogonal nanospray source tion is calculated by multiplying the peak Solvent tray and nanocolumn Microdegasser capacity of the single separation steps [17]. Nanoflow pump By combining two different LC techniques with individual peak capacities Solvent tray Micro-WPS between 50 and 100, it will theoretically Microdegasser MSD ion trap XCT Binary pump be possible to achieve total capacities of Cooler 250010,000 peaks. Micro valve and holder Two different approaches have been B addressed in order to separate and resolve Binary pump Autosampler complex protein expression patterns: the multidimensional separation of intact Nanobore proteins [18,19] and the combination of column different techniques adapted for separation at the peptide level [16]. The comWaste plete workflow for the latter approach MS detection can be more easily automated since the digestion of the complete protein conNanoflow Enrichment tent of a sample takes place at the beginpump column ning of the workflow. In addition, this approach is also more suitable to mini- C Waste mize nonspecific adsorption to separation devices and tubings, which is much Enrichment Injector less pronounced with peptides than with column Nanoflow pump B intact proteins. The initial complexity of the starting sample, however, is RP analysis increased significantly by increasing the Nanobore total amount of different compounds by Ion trap MS Binary pump column one to two orders of magnitude. For the separation of complex peptide mixtures, Figure 1. (A) Basic nano-LC/MS system for 1D enrichment LC/MS and online 2D LC/MS. (B) Valve configuration of this LC/MS system. (C) Principle of 1D enrichment LC/MS. the combination of several orthogonal LC: Liquid chromatography; MS: Mass spectrometry; MSD: Mass selective detector; RP: Reversed phase; techniques have been used: XCT: X-ray computed tomography.

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2D-LC/MS techniques for the identification of proteins

large proteomes, as well as identifying A a subset of proteins expressed under 1st dimension special conditions [33,34]. Desalt Waste salt gradient in steps In contrast to this well-established SCX online methodology, offline 2D-LCcolumn Enrichment MS/MS significantly increases chroNanoflow column pump matographic resolution of highly com2nd dimension plex proteome samples. This improveInjector RP analysis ment is attributed to continuous Nanobore linear gradient SCX-chromatography Ion trap MS Binary pump column in the first dimension without intermitting RP-chromatography intervals. As a consequence, a higher resoB lution and peak capacity and therefore Second pump a higher number of identified proteins is associated with the offline 2D-LC1100 series microWaste well plate autosampler MS/MS approach. For fraction collecNano Reversed phase pump tion in the first dimension (SCX) a trapping column microfraction collection device must be used. In the second dimension nano-RP-LC/MS is applied to the separation of the eluent obtained from Column Reversed phase the first dimension. The offline Detection switch column approach has several advantages over Strong cation the online SCX methodology: 1100 series column exchange compartment Peptide separation is superior in a column linear gradient More organic solvent can be used to Waste improve chromatography Figure 2. (A) Principle of 2D-LC. (B) Valve switching configuration of the LC modules and localization More peptide fractions can be of the columns. collected LC: Liquid chromatography; MS: Mass spectrometry; RP: Reversed phase; SCX: Strong cation exchange. Chromatography and corresponding fraction collection can be more interactively optimized Thermostatted column compartment with micro for the individual sample by using the ultraviolet trace of the two-position/six-port valve chromatogram and adjusting fraction width accordingly Diode-array detection (DAD) with 500- or 80-nl flow cell Fractions can be reanalyzed or chemically modified [30] Microfraction collector with thermostat The offline SCX technique has been very successfully used to elucidate the proteomes from yeast [30] and human serum [35]. Software
Results & discussions Experimental equipment
Nanoflow proteomics solution from Agilent Technologies [101,102]:

ChemStation A10.02 Spectrum Mill MS Proteomics Workbench [104] Data processing was performed with Spectrum Mill software operated on a dual 2.4 GHz Xeon workstation with Windows 2000 Server operating system. MS/MS data were searched against the species subset of the National Center for Biotechnology Information nonredundant database. Hits with a high score were validated automatically, medium score hits were inspected and validated manually. Remaining spectra were subject to a PTM search following validation in an iterative process. The Spectrum Mill scoring provides a measure of match quality. It includes mass analyzer specific rule sets and is based on factors such as the fragment ions present, sequence tag length and peak intensity.

Nanoflow pump with microvacuum degasser Thermostatted microwell-plate autosampler Micro two-position/six-port or two-position/ten-port switching valve box Quaternary, binary or capillary pump LC/mass selective detector (MSD) Trap XCT with orthogonal nanospray ion source
Microfraction collection system from Agilent Technologies [103]:

Capillary pump with microvacuum degasser Thermostatted microwell-plate autosampler

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Ngele, Vollmer, Hrth & Vad

Peptidoglycan synthesis 4 Transfer RNA amino acid synthesis 14 Amino acid synthesis and metabolism 28 Outer membrane 16

Ribosomal proteins 20 Carbohydrate Transporter/binding proteins 32 metabolism and Translation 5 synthesis 65 Other cytosolic proteins 38 DNA replication, transcription 47 Inner membrane 13 Unknown function proteins 76 Folding, stress, proteases 22 Nucleotide metabolism 21 Fatty/phosphatidic acid metabolism 25 Periplasmic proteins 35
Figure 3. Number of proteins identified in an Escherichia coli whole-cell lysate according to their metabolic pathway or cell localization.

from the nanoflow pump. The peptides are then separated on the nanobore column prior to their mass spectrometric analysis. This instrument is capable of analyzing samples containing digested proteins from, for example, a gel spot, a small gel band or a fraction from the first dimension of an offline 2D-LC system (vide infra). This basic system can be enhanced with an additional SCX column, a high-precision capillary pump, a micro ten-port valve or included in an offline 2D-LC system for 2D-LC applications of highest performance.
Injected salt step solution approach of online 2D-LC

The principle of the 2D nano-LC is illustrated in FIGURE 2A [32]. The SCX column was directly connected Basic 1D enrichment nano-LC/MS system to the injection needle seat of the microwell-plate autosampler The basic setup of the LC/MS instrument is illustrated in and mobile phase was pumped from the binary pump through an autosampler and SCX column. The column outlet was conFIGURE 1A. The principle of the 1D enrichment LC and the according valve configuration is shown in FIGURE 1B. In general, nected to the two-position/six-port valve located in the autosamthe tryptic peptides are injected and concentrated on a C18 pler, which allows bypassing of the SCX column from the mainenrichment column, which is connected to a micro six-port flow. Flowthrough of nonbinding peptides was directed to the valve. After washing, this column is switched into the nanoflow two-position/six-port valve in the column compartment and path and the peptides are eluted with the gradient delivered enriched on a short C18 trapping column, which was mounted in between two ports of this valve (FIGURE 2B). During the first dimension, the enrichment A 1st dimension 2nd dimension column was inline with the SCX column and the flowthrough of the enrichment colNanoflow SCX column LC pump umn was directed to waste, which allowed Enrichment washing off of salts and other nonbinding Enrichment column Enrichment Injector Ion trap contaminants that would have been disadcolumn 1 column 2 cycle vantageous for later MS analysis. Elution Nanobore from SCX is obtained stepwise by injecting Capillary column pump increasing concentrations of salt solution from the autosampler. After each salt injecWaste tion the enrichment column was switched in the second flow path where mobile Enrichment column 2 B phase was directed from the nanoflow pump through the valve to the RP nanocolumn. The outlet of the column was Waste Nanoflow pump directly connected to the sprayer needle in the orthogonal ion source of the ion trap Capillary pump Column autosampler switch MS instrument. Switching the enrichment column into the nanoflow path results in a reversed flow trough. Increasing the conSCX column Nanobore column centration of organic solvent eluted the and MS detection sample, which was concentrated in the Enrichment column 1 enrichment column and further separation was achieved on the analytical RP nanoFigure 4. (A) Principle of online 2D-LC with semicontinuous gradient using two revolving enrichment column. In the following procedure the columns as an interface between the two dimensions. (B) Valve configuration for online 2D-LC. trapping column was switched back into Chromatography with semicontinuous gradient. the solvent path of the SCX column for the LC: Liquid chromatography; MS: Mass spectrometry; SCX: Strong cation exchange. next elution step.
HPLC systems for online & offline 2D-LC

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2D-LC/MS techniques for the identification of proteins

100 90 80 70
250-500 mM

500mM

Fractions:
Unbound 0-12.5 mM 12.5-25 mM 25-37.5 mM 37.5-50 mM 50-75 mM 75-100 mM 100-150 mM 150-250 mM 250-500 mM 500mM

% Solvent B

60 50 40 30 20 10 0
0-12.5 mM 12.5-25 mM 25-37.5 mM 37.5-50 mM 100-150 mM 75-100 mM 50-75 mM

150-250 mM

Figure 4. (C) Online 2D semicontinuous gradient liquid chromatography methodology showing gradients for SCX, reversed phase separation and valve switching points. SCX: Strong cation exchange.

With this online 2D nano-LC/MS system one can analyze samples of medium complexity, such as larger gel bands, subproteomes or upregulated proteins in a whole-cell digest. As an example, Escherichia coli cultures were treated with different environmental conditions such as heat shock [34] or different nutrition conditions [33]. In the experiment with the heat-shocktreated E. coli culture, prominent, upregulated heat-shock proteins were identified in a whole-cell lysate compared with an untreated control group. In the second experiment, a whole-cell lysate of an E. coli strain grown on glucose was compared with an E. coli strain grown on lactose. In the lactose-grown bacteria, upregulation of metabolic enzymes necessary for the lactose degradation were identified in a background of hundreds of other proteins. The final result of this experiment is shown in FIGURE 3. This cell sketch summarizes more than 400 identified proteins according to their metabolic pathway or cell compartment.
Improved online 2D-LC approach with a semicontinuous gradient

The most frequently used approach for the separation of peptides from protein digests in complex proteomic applications by HPLC is 2D nano-LC/MS using a SCX column in the first dimension and a RP column in the second dimension. In the most frequently used method, the sample peptides bound on the SCX columns were eluted with injected salt solution plugs of increasing concentration. However, the injected volume of salt solution is not optimized to the SCX column and the SCX column is working far away from its optimum state. The unoptimized state of this method gives reason for the distribution of the peptides over more than one fraction, which can dilute them below their detection level or suppress their ionization in nanoelectrospray by higher abundant peptides in the MS analysis.

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100

150

200

250

300

350

Time (mins) Salt step elution Nanoflow elution gradient Injection point SCX mainpath SCX bypass Enrichment column exchange

To overcome these limitations, an improved method for online 2D-LC was developed. In this method, the optimized semicontinuous salt solution gradient for the elution of the peptides from the SCX column is delivered very precisely with a capillary pump, and the SCX column was always kept under conditions very close to the optimum state. The eluted peptides are trapped rotatory on two enrichment columns and are subjected to RP separation followed by MS/MS analysis. The principle of this method is sketched in FIGURE 4A. At the starting point of each RP analysis cycle the charged enrichment column is exchanged against the empty column by a switch of the micro ten-port valve (FIGURE 4B). For the chromatographic separation in the first and second dimension, it is necessary to set up two different methods, one for the SCX chromatography and another one for the RP separation (FIGURE 4C). The salt solution gradient is developed for 15 min in each step and prior to the washing step the SCX column is switched to bypass with the micro six-port valve in the autosampler to retain the optimum state condition (the switching points of the valves are included as bullet points in FIGURE 4B). Therefore, each step contributes to a quasi-linear salt gradient on the SCX column. To get a good separation for the majority of peptides eluting at lower salt concentrations, the slope is shallower in this area and steeper in the area of higher salt concentrations. To show the full performance of this method, a complex tryptic digest of the yeast proteome was analyzed. The comparison of the results obtained with this new semicontinuous gradient method to the currently used step injection method showed an increase in the number of reliable identified proteins of more than 20%.
Offline 2D-LC for high-performance separations

In order to identify proteins from a highly complex mixture of 5 103 to 5 104 compounds with a dynamic range of at least

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After reinjection, the peptides from each fraction are concentrated on a short C18 enrichment column located between two Solvent tray TCC + micro 6-port valve Microdegasser ports of the microvalve. The enrichment Capillary pump DAD column was then switched into the nanoMicrofraction collector Micro-WPS flow path, the concentrated peptides were eluted, further separated by a gradient with Cooler Cooler increasing organic solvent on an analytical 2nd dimension: nano-LC/MS system nanobore column and sprayed directly into Solvent tray Orthogonal nanospray the MS. The typical workflow for an offline source and nanocolumn Microdegasser 2D-LC proteome analysis is depicted in Nanoflow pump FIGURE 5B. The working principle of the Micro-WPS nano-LC/MS system in 1D enrichment Solvent tray MSD ion trap XCT mode was previously explained. Cooler Binary pump For the comparison of the online 2DMicro 6-port valve LC/MS method working with the injected salt solution plugs of increasing B Loading concentration and the offline 2D-LC pump method working with a real continuous salt solution gradient in the first dimen1st dimension sion, a whole-cell lysate from yeast (S. cerTransfer of Injector fractions evisiae) was analyzed. Sample preparation Micro was performed according to published fraction Nano LC collecton protocols using cooled resuspended Capillary Enrichment pump Fractionation pump lyophilized yeast cells (Sigma GmbH, in column with a continuous 50 mM AmBic/8M urea) in a bead beater 2nd dimension salt gradient Desalt (BioSpec Products) with 0.5-mm glass SCX Nanobore Ion trap MS Waste beads [33]. Shortly, proteins were reduced Injector column column with DTT, 37C, alkylated with iodoaAnalysis cetamide, followed by ultrafiltration for Figure 5. (A) Offline 2D-LC/MS system containing a microfraction collection system and a nano-LC/MS buffer exchange (50 mM AmBic). Tryptic system. (B) Working principle of the 2D-LC/MS system. digest was performed using TPCK trypsin DAD: Diode-array detection; LC: Liquid chromatography; MS: Mass spectrometry; (Pierce). Digested peptides were lyophiSCX: Strong cation exchange. lized, washed in deionized water followed 105, it will be crucial to develop technologies with extremely good by a second lyophilization using a SpeedVac (Bachofer). Finally resolving power on one hand and with extraordinary sensitivity the sample was dissolved in buffer A prior to analysis. For on the other hand. To improve the multidimensional LC, an HPLC separation, sample aliquots were taken from the same offline 2D-LC approach was developed and compared with the frozen sample batch. All chemicals and bioreagents used in this online 2D-LC approaches discussed thus far. The first dimension study were obtained from Sigma GmbH. The 2D-LC separation was performed with identical buffers. starts with the elution of tryptic peptides from a SCX column with a real continuous linear salt gradient and the subsequent col- While the first dimension differed in respect to injected salt lection of fractions in the l range with the microfraction collector plugs versus linear gradient, the RP runs for the second separation dimension were identical for all three methodologies. Con(FIGURE 5A). In order to collect small volumes in the lower l range reliably, a unique collection principle has been developed. At the cerning the SCX dimension, injected salt plugs corresponded to start of fraction collection, the droplet being formed is precisely the concentration ranges of the fractions taken in the offline deposited at the bottom of the well. During the entire collection technique. Analysis times were similar with all three systems. The overall outcome of the analysis demonstrated that more process, the fraction collector ensures that the droplet forming at the capillary tip is in constant contact with the surface of the proteins with higher score and sequence coverage could be deposited liquid. As the well is filled the capillary tip moves con- identified with the offline 2D-LC approach. The top score protinuously upwards, keeping constant contact with the solvent sur- teins are shown in TABLE 1. To demonstrate the full resolution face. When fraction collection is finished the capillary tip moves performance of offline 2D-LC, the distribution of the peptides quickly upwards, ensuring that the droplet currently being over the collected fractions from the first dimension was disformed at the capillary tip is delivered to the well. The well-plate, played (TABLE 2). This distribution clearly shows that most of the containing all collected fractions, was then transferred directly to identified tryptic peptides of the protein phosphopyruvate the second dimensionthe nanoflow RP-LC system (FIGURE 1 & 5A). hydratase are only found in one fraction.
A 1st dimension: Microfraction collection system

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2D-LC/MS techniques for the identification of proteins

Table 1. Comparison of the highest protein scores for online (left columns) and offline (right columns) 2D liquid chromatography/mass spectrometry analysis of the yeast cell lysate.
Protein name
Phosphopyruvate hydratase Phosphoglycerate kinase Pyruvate decarboxylase Glucose kinase Translation elongation factor eEF-1 Citrate synthase Pyruvate kinase Hexokinase A Alcohol dehydrogenase 1 Phosphoglycerate mutase 1 Eukaryotic translation initiation factor 5A-2 Phosphogluconate dehydrogenase Glycerol-3-phosphate dehydrogenase Superoxide dismutase 10 8 8 1 7 1 6 4 5 4 3 2 1 2

Peptides
12 10 8 6 7 5 5 4 3 4 4 3 3 2 45 52 21 2 76 3 16 16 39 16 7 7 2 2

Spectra
27 32 14 8 17 7 8 6 9 8 5 5 5 3

Coverage
36 25 24 4 23 3 21 13 16 23 22 22 5 27 42 32 14 19 23 13 19 14 15 13 23 23 11 27 150.46 123.75 114.54 16.33 98.57 16.97 81.53 65.13 62.63 55.54 47.39 47.39 16.79 24.70

Score
161.50 150.45 112.92 97.77 93.12 73.11 71.66 64.03 42.49 55.24 56.07 56.07 41.15 30.14

Compared with the online method, the offline method yields better resolution, which in turn results in greater sequence coverage and therefore higher scores in cases where the same proteins are identified by both methods. Higher scores are emphasized when the difference is >5%.

Conclusion

In this review, several HPLC instrument setups and their system internal workflow for multidimensional analysis of proteome samples of different complexity are described. The basic system described is working in a 1D enrichment mode. This system is capable of handling complex proteome samples sufficient for the analysis of digested proteins from larger gel spots, gel bands, protein complexes or smaller subproteome samples. The basic concept of loading a potentially contaminated in-gel digest sample on an enrichment column with a subsequent washing step followed by the separation on a nanobore column and MS analysis is used in all more complex multidimensional systems described here. The most simple instrument setup for online 2D-LC/MS system works with the principle of elution of the tryptic peptides from the first dimension SCX column with injected salt solution plugs of increasing concentration. The eluted peptides are again trapped and introduced into the nanoflow path for separation and analysis. The great advantage of the system is a robust and fully automated separation; it is able to perform the complete analysis in an unattended fashion. The method is easy to set up and composed of a sequence of identical runs differing only in the concentration of the injected salt plugs. The disadvantage of this system is that the SCX column is working far away from its optimum under these conditions. Resolution and the number of salt steps are rather limited with this approach.

To overcome these drawbacks in an online 2D-LC system, a more complex instrument setup capable to work with a pumped semicontinuous gradient was developed. This enhanced system enables the investigator to obtain more resolution out of a given sample without compromising automation. However, setting up multiple SCX runs timely matched with multiple RP runs all included in a single 2D-LC program requires good knowledge and expertise in the LC software. For the analysis of proteome samples of highest complexity, such as whole-cell proteome digests or serum analyses, a HPLC instrument setup for offline 2D/MS was developed. This system separates the two dimensions in two different HPLC systems. In the first system, the SCX separation is performed with a real linear continuous gradient for highest resolution and the eluent is collected in small l fractions. These fractions are then transferred to the nano-LC/MS system responsible for RP separation and MS analysis of the peptides. This system operates in the described enrichment mode. The advantages of the system are high resolution, user-defined fraction width and number, easy optimization by using ultraviolet light detection, chemical manipulation possibility and a reanalysis option. In order to compare the performance of these systems under similar conditions, the same amount from a whole-cell digest from yeast (S. cerevisiae) was injected onto the same SCX column. In addition, injected salt steps and fractions obtained from the linear SCX gradient separation covered the same salt concentration ranges. As a result, 101 proteins could be identified from the

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Table 2. Distribution of phosphopyruvate hydratase tryptic peptides over the collected fractions in an offline 2D liquid chromatography/mass spectrometry experiment.
Sequence m/z measure
597.49 790.13

MH+ match 1-3 4 5 6

1789.84 1578.80 14.90 10.83 13.71 9.79 14.19 12.75 14.25

Fraction score 7 8
10.60

10

11 12-13

(K)AAQDSFAAGWGVMVSHR(S) (K)AVDDFLISLDGTANK(S)

(R)GNPTVEVELTTEK(G) (R)IEEELGDNAVFAGENFHHGDKL(-) (K)IGLDCASSEFFK(D) (K)NVNDVIAPAFVK(A)

708.94 814.69 687.51 644.07

1416.72 2441.14 1373.64 1286.71

18.84 12.43 12.34 13.21 11.83 9.09 10.00 15.50 12.74 9.62 13.90 18.35 16.79 12.20 9.23 13.19 16.50 12.30 12.49

(K)NVPLYKHLADLSK(S) (R)SGETEDTFIADLVVGLR(T) (R)SIVPSGASTGVHEALEMR(D)

749.95 911.52 921.50

1497.84 1821.92 1840.92

(K)TAGIQIVADDLTVTNPK(R)

878.37

1755.95

(K)VNQIGTLSESIK(A) (K)WLTGPQLADLYHSLMK(R)
m/z: Mass/charge ratio.

645.09 625.09

1288.71 1827.97

Table 3. Comparison of the online 2D-LC methods.

Method
Identified proteins Assigned peptides SCX resolution Automation Complexity of setup Investment Effort Access to SCX fraction Flexibility
LC: Liquid chromatography; SCX: Strong cation exchange.

Online 2D-LC with injected salt steps

101 179 Low High Medium Low Medium No Low

Online 2D-LC with pumped semicontinuous salt gradient

122 207 High High High Medium Medium No Low

Offline 2D-LC with pumped linear salt gradient

144 269 Very high Medium Medium High Medium Yes High

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system working with the injection method, 122 proteins with the system working with the semicontinuous salt solution gradient method and 144 from the offline 2D-LC system working with the real continuous salt solution gradient in the first dimension. The comparison of the number of identified proteins, assigned peptides and some other aspects is outlined in TABLE 3. The goal of the comparison was not to identify a maximum of proteins from yeast but to compare the three systems under similar conditions and similar analysis times. Literature and our own observations give evidence that by extending the linear gradient and by increasing fraction number concurrently with decreasing fraction width, the number of identified proteins increases dramatically [30,35]. This, however, stands in favor of the 2D-LC offline methodology, since the number of salt plugs that can be injected in the online technique are limited and can never be as fine tuned as is the case in a linear gradient with fraction collection.
Expert opinion

2D-PAGE due to, for example, very hydrophobic membrane proteins (because of the importance of these protein classes in the development of new pharmaceutical compounds). However, multidimensional LC/MS will not eventually replace the 2D-gel techniques because of their high resolving power. Therefore, both separation techniques will contribute complementarily to the progress in proteomic research and drug development. In comparison with online 2D-LC/MS, the offline 2D-LC/MS approach provides higher resolution and increased flexibility. This will promote the offline 2D-LC methodology as the best choice for future needs in proteomic separations.
Five-year view

Today, 2D-LC/MS techniques are part of the repertoire in the analysis of proteome samples. There are different approaches available, which can be chosen in accordance to the complexity of the proteome sample. These LC techniques will gain importance in the analysis of samples that are difficult to analyze with Key issues

Starting with lab-on-a-chip technology using the Bioanalyzer, Agilent is continuing to build on this miniaturization technique/knowledge. Currently, Agilent Labs, Agilents research facility, is investigating new ways to simplify proteomic work to make techniques such as LC/MS and LC-MS/MS more user friendly. This new approach is integrating the LC separation part into a chip environment and is simplifying the connection to the mass spectrometer. The customer will benefit from this new chip LC technology it will provide ease of use, robustness and high performance and will open up complicated proteomic applications to the less-experienced user.

An introduction to different approaches of multidimensional high-performance liquid chromatography (HPLC) is given. Equipment and software for multidimensional nanoliquid chromatography/mass spectrometry (LC/MS) for proteomic applications is described. The basic HPLC/MS system and its internal workflow for multidimensional nano-LC/MS is described in detail. A nano-HPLC/MS system for improved online 2D-LC/MS is described. An HPLC/MS system for offline multidimensional LC, containing a microfraction collection LC system and a nano-LC/MS system, for the analysis of proteome samples of highest complexity, is described. The performance of the online and offline LC/MS systems is compared with the obtained results from the analysis of a complex sample. The multidimensional LC technique is discussed in the context of existing techniques. An outlook into the future development of chip LC technology is given.

References
1

Pennington SR, Dunn MJ. Proteomics: from Protein Sequence to Function. BIOSScientific Publishers Ltd., Oxford, UK (2001). Wilkens MR, Williams KL, Appel RD, Hochstrasser DF. Proteome research. In: New Frontiers in Functional Genomics. Springer Verlag, Berlin, Germany (1997). Phizicky E, Bastiaens PIH, Zhu H, Snyder M, Fields S. Protein analysis on a proteomic scale. Nature 422, 208215 (2003).

Pandey A, Mann M. Proteomics to study genes and genomes. Nature 405, 837846 (2000). Tyers M, Mann M. From genomics to proteomics. Nature 422, 193197 (2003). Rabilloud T. Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2, 310 (2002). OFarrell PH. High resolution twodimensional electrophoresis of proteins. J. Biol. Chem. 250, 40074021 (1975).

Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26, 231243 (1975). Goerg A, Obermaier C, Boguth G et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 10371053 (2000). Lopez MF. Better approaches to finding the needle in a haystack: Optimizing

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45

Ngele, Vollmer, Hrth & Vad

proteome analysis through automation. Electrophoresis 21, 10821093 (2000).

11

23

Patton WF. Detection technologies in proteome analysis. J. Chromatogr. B 771, 331 (2002). Liu H, Lin D, Yates JR III. Multidimensional separations for protein/peptide analysis in the post-genomic era. BioTechniques 32, 898911 (2002). Wu CC, Yates JR III. The application of mass spectrometry to membrane proteomics. Nature Biotechnol. 21, 262267 (2003). Wu CC, MacCoss MJ, Howell KE, Yates JR III. A method for the comprehensive proteomic analysis of membrane proteins. Nature Biotechnol. 21, 532538 (2003) Hanash S. Disease proteomics. Nature 422, 226232 (2003). Wang H, Hanash S. Multi-dimensional liquid phase based separations in proteomics. J. Chromatogr. B 787, 1118 (2003). Giddings JC. J. High Resolut. Chromatogr. 62, 978 (1990). Lubman DM, Kachman MT, Wang H et al. Two-dimensional liquid separations mass mapping of proteins from human cancer cell lysates. J. Chromatogr. B 782, 183196 (2002). Zheng S, Schneider KA, Barder TJ, Lubman DM. Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 E.coli. BioTechniques 35, 12021212 (2003). Larmann JP, Lemmo AV, Moore AWJ, Jorgenson JW. Two-dimensional separations of peptides and proteins by comprehensive liquid chromatographycapillary electrophoresis. Electrophoresis 14, 439447 (1993). Issaq HJ, Chan KC, Janini GM, Muschik GM. A simple two-dimensional high performance liquid chromatography/high performance capillary electrophoresis setup for the separation of complex mixtures. Electrophoresis 20, 15331537 (1999). Opiteck GJ, Lewis KC, Jorgenson JW, Anderegg RJ. Comprehensive on-line LC/LC/MS of protein. Anal. Chem. 69, 15181524 (1997).
28 27 24

Wolter D, Washburn MP, Yates JR III. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73, 56835690 (2001). Davis MT, Beierle J, Bures ET et al. Automated LC/LC-MS/MS platform using binary ion-exchange and gradient reversedphase chromatography for improved proteomic analyses. J. Chromatogr. B 752, 281291 (2001). Mawuenyega KG, Kaji H, Yamauci Y et al. Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography-tandem mass spectromtry. J. Proteome Res. 2, 2335 (2003) Giggs L, Sioma C, Regnier FE. Automated signature peptide approach for proteomics. J. Chromatogr. A 924, 359368 (2001). Wang S, Zhang X, Regnier FE. Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates. J. Chromatogr. A 949, 153162 (2002). Adamczyk M, Gebler JC, Wu J. Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry. Rapid Commun. Mass Spectrom. 15, 14811488 (2001). Washburn MP, Wolters D, Yates JR III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnol. 19, 242247 (2001). Peng J, Elias JE, Thoreen CC, Licklider LJ, Gygi SP. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for largescale protein analysis : The yeast proteome. J. Proteome Res. 2, 4350 (2003). Florens L, Washburn MP, Raine JD et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520526 (2002). Ngele E, Vollmer M, Hrth P. Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics. J. Chromatogr. A 1009, 197205 (2003).

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12

Vollmer M, Ngele E, Hrth P. Differential proteome analysis: twodimensional nano-LC/MS of E. coli proteome grown on different carbon sources. J. Biomol. Tech. 14, 128135 (2003). Ngele E, Vollmer M, Hrth P. Proteome profiling in E. coli: Effect of heat-shock conditions on protein expression pattern. LC/GC Europe 16, 641647 (2003). Adkins JN, Varnum SM, Auberry KJ et al. Towards a human blood proteome. Mol. Cell. Proteomics 1(12), 947955 (2002).

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Affiliations
Edgar Ngele Agilent Technologies R&D & and Marketing GmbH & Co. KG, Hewlett-Packard-Str. 8, Waldbronn, Germany Tel.: +49 724 360 2663 edgar_naegele@agilent.com Martin Vollmer Agilent Technologies R&D and Marketing GmbH & Co. KG, Hewlett-Packard-Str. 8, Waldbronn, Germany Tel.: + 49 602 630 martin_vollmer@agilent.com Patric Hrth Agilent Technologies R&D and Marketing GmbH & Co. KG, Hewlett-Packard-Str. 8, Waldbronn, Germany Tel.: +49 724 360 2681 patric_hoerth@agilent.com Cornelia Vad Agilent Technologies R&D and Marketing GmbH & Co. KG, Hewlett-Packard-Str. 8, 76337 Waldbronn, Germany Tel.: +49 724 360 2854 cornelia_vad@agilent.com

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Agilent publication number 5989-1396EN

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Expert Rev. Proteomics 1(1), (2004)