Chromatographic Techniques

for the Separation of Peptides:

Application to Proteomics
2007, 65, 511518

Srinubabu Gedela&, Narasimha Rao Medicherla

Center for Biotechnology, Department of Chemical Engineering, Andhra University, Visakhapatnam 530003, India;
E-Mail: srinubabuau6@gmail.com

Received: 26 December 2006 / Revised: 29 January 2007 / Accepted: 19 February 2007

Online publication: 5 April 2007

Abstract
The greatest challenge for proteomics is the inherently complex nature of cellular proteomes as
they are highly dynamic entities. High performance liquid chromatography is an indispensable
tool in proteomics research, providing high-speed, high sensitivity separation and good resolution of proteins and peptides. Chromatographic sciences have played an animated, bustling
and critical role in many fields, the next challenging analytical project for the chromatographic
scientists is in the area of proteomics. Which type of analysis best determines the optimal
separation technique for any proteomic study? The aim of this review is to outline the different
chromatographic strategies that have been employed for analysis of complex mixtures of proteins/peptides, highlighting the role of liquid chromatography coupled to mass spectrometry.

Keywords
Column liquid chromatographymass spectrometry
Peptide separation
Proteomics

Introduction
A protein may be dened as a set of
amino acids arranged in a specic sequence to yield a dened activity or
property. Genes are the known DNA
stretches that code for a macromolecular
protein involved in various processes
necessary for normal growth and development of a living organism.
However the functions of particular
proteins at the cellular or sub cellular

Review
DOI: 10.1365/s10337-007-0215-9
0009-5893/07/05

levels can be done only after deciphering

all the genes in a particular living being
and this marks the development of the
next engender technologies. The full draft
of the human genome map was presented
by Francis Collins, Director of the Human Genome project (HGP), and Craig
Venter, Chairman of Celera Genomics,
on 26th June 2000, ve years ahead of
schedule. The HGP started in the 1990s
and was funded by the US National
Institute of Health (NIH) and partly by
the food and drug administration (FDA)
as biologys rst large science project
planned to be completed in 15 years with

an outlay of three billion dollars. It was

an international collaborative eort
involving 15 countries and 250 laboratories around the globe. The aim was to
create a map of the entire human genes
by decoding three billion base pairs.
The project was completed ahead of
the schedule as several new methods, such
as the laser beam decoding machine of
Michael Hunkapiller in 1998 and the
shotgun sequencing technique of Craig
Venter in 1999 [1], were developed. Now
in the post genomic era, HGP is speculated to engage in the next step, i.e.
Emotional Genomics [2]. The human
genome consists of approximately
30,00050,000 genes that yield more than
100,000 different proteins owing to
alternative splicing [3] generating a large
number of various splice variants and a
range of post-tranlational modications,
which occur in a contingent manner
resulting in a plethora of proteins and
their varied forms in the range of 100300
fold variants is in the order of 108109 [4].
The analytical methods required need to
be highly sensitive and quantitative. One
distinctive advantage of proteomics is the
potential of detecting changes that occur
after the messenger RNA step thus giving
a quantitative analysis of protein expression proles [5].
Proteomics deals with the study of
proteins, their structure, localizations,
posttranslational modications, functions and interaction with other proteins.
The mapping of protein structure function holds the key to a better understanding of cellular functions under both

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2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

511

normal and disease states, which is critical for modern drug discovery. Proteome
analysis allows the discovery of the constituents of proteomes; typically intact
proteins are digested into peptides and
fractionated using a variety of chromatographic techniques prior to their
actual identication using some form of
mass spectrometry and/or gel electrophoresis. In the last few years signicant
research has been carried out to develop
chromatographic methods to enable the
resolution and identication of all the
peptides in a given proteome. This task is
not trivial because, for example, a serum
proteome may contain up to 30,000 proteins, which with a dynamic concentration range of up to 1010, may result in
400,00600,000 peptides in a digest [6, 7].
Many attempts have made to resolve
such complex protein/peptide mixtures.
However, no method presently exists that
can be used to separate, detect and
quantify all proteins within a given proteome.
The aim of this review is to give an
account of the dierent techniques that
have been employed for the separation of
complex mixtures of proteins/peptides
and highlight the critical role of mass
spectrometric detection.

Analytical Techniques
A proteomic analysis of a sample usually
consists of four steps: extraction of the
proteins from the sample, their separation, detection and nally identication/
analysis of individual separated proteins
[8]. The type of analysis being undertaken
depends on the required objective. Does
the study require the separation and
analysis of a single specic protein, a
group of proteins, or all the proteins in a
cell or tissue? The conundrum, therefore
denes which technique [gel electrophoresis (GE), high performance liquid
chromatography (HPLC), capillary electrophoresis (CE)], a combination of
techniques (multidimensional), or selective separation (isoelectric focusing,
afnity, ion exchange, size exclusion, etc.)
should be used.
Proteins are linear polymers of amino
acids and the following four structural
features distinguish them from each other
and can be used to separate them.
1. Charge state
2. Size and shape

512

3. Polypeptide chain length

4. Polypeptide hydrophobicity
Gel electrophoresis (GE) in conjunction with advanced mass spectrometric
techniques, has facilitated the rapid
characterization of thousands of proteins
on a single polyacrylamide gel plate. The
ability of this technique to separate
thousands of proteins in a cell or tissue,
including their posttranslationally modied forms, has resulted in it becoming the
major separation technique in protein
analysis and thus is well suited for the
global analysis of protein expression in a
living being. This, together with the newly
developed activity-based proling approaches that target dierent classes of
proteins, has now allowed the study of
protein functions based on their intrinsic
enzymatic activities [9, 10]. However GE
suffers from a number of long standing
problems including low throughput, a
limited dynamic detection range, poor
reproducibility, low sensitivity as well as
its difculties in analyzing hydrophobic,
small and very basic or acidic proteins.
Incremental enhancements in GE,
including the use of sensitive staining
methods, higher resolving gels and sample fractionation prior to GE, have reduced but not eliminated, some of these
problems [11, 12]. Differential gel electrophoresis [13] and a multiplexed proteomic approach [14] are some other recent
developments related to GE.

Peptide Separation
In the past, it has been demonstrated that
peptides can be analyzed by mass spectrometry requiring very little or no
manipulation of the sample, just by
placing the tissues directly on the
MALDI target plate and applying the
matrix solution [15, 16] or, in the case of
ESI, by a brief extraction in the spraying
solvent [1517]. Currently, chromatographical separations at ow rates as low
as 20 nL min-1 using columns with
internal diameters down to 15 lm [18]
have resulted in higher sensitivity.

Affinity Chromatography
Anity chromatography represents one
of the most powerful fractionation techniques for purication of macromolecules

Chromatographia 2007, 65, May (No. 9/10)

including modied proteins [19, 20]. It

has been used to give several fold purication in a single step, usually with high
recovery. Despite the potential value of
afnity chromatography, it is limited by
the availability of specic ligands for each
protein target. This limitation may be
overcome by screening combinational libraries or computer modeling. Recently
Mondal and Gupta [21] have reviewed
the afnity concept in bio-separations.

Immobilized Metal Ion Affinity

Chromatography (IMAC)
IMAC is based on the interaction between molecules in solution and metal
ions immobilized on a solid support. The
molecules are separated according to
their anity for chelated metal ions,
which depends on the coordination between the chelated metal ion and electron
donor groups forming the ligand. Two
characteristics of the metal-ligand bonds
can be used for the successful separation
of dierent ligands. Firstly, the strength
of the metal-ligand bond varies from ligand to ligand and secondly, binding
between the immobilized metal ions and
the ligand is reversible. Hence, elution
can be carried out by changing the conditions thereby breaking the metal-ligand
bonds. Three dierent elution principles
are used in IMAC, such as a competitive
elution, stripping elution and pH adjustment. Low cost of stationary phase, high
binding capacity and relative high specicity of protein and metal interaction are
the key advantages of IMAC. IMAC has
been widely used in the purication of
peptides [2226] and the application of
IMAC has been extended to the purication of viral vectors for use in gene
therapy application [27, 28]. Recently
Jiand et al. [29] applied this technique for
the purication of herpes simplex virus
type 1 (HSV-1) gene therapy vectors.
Stationary phases with improved stability are still the subject of intense
research in the eld of liquid chromatography, to yield more ecient and
faster analysis. Various approaches have
been taken to enhance the stability of
reversed phases by using inorganic metal
oxides such as zirconia [30], alumina [31],
and titania [32, 33]. Recently Yue et al.
[34] described an integrated glass microdevice for proteomics, which directly
couples proteolysis with afnity selection.

Review

They reported the peptide capture from

standard solutions and protein digests on
a simple microdevice using IMAC beads
packed into a microuidic channel.

Ion Exchange
Chromatography
The use of ion-exchange chromatography
is based on the theory that each protein
has its own unique physiochemical
properties such as ion changes [35, 36].
Correct mobile phase selection has signicantly progressed high-performance
ion-exchange fractionation of proteins
[37]. The pH and salt content of the eluent affects chromatographic behavior on
both strong and weak ion-exchange columns. Although anion-exchange chromatography has been widely used for
protein separation, cation-exchange
chromatography is also a useful separation method, as approximately one-third
of all proteins reported in the literature
have an isoelectric point (pI) sufciently
high to be resolved by cation-exchange
chromatography [38].

Size-Exclusion
Chromatography
The use of size-exclusion chromatography is based on the theory that each
protein has its own unique structure and
molecular size, which is suitable, in particular where biologically active proteins
(e.g. enzymes, hormones and antibodies)
are processed. Full recovery of activity
after chromatography may be achieved
and current size-exclusion columns allow
fractionation from Mr 10,0001,000,000
[39]. Extremely basic or hydrophobic
proteins may not exhibit true size-exclusion character, as the columns tend to
have slight hydrophobicity and anionic
character. Thus a combination of sizeexclusion chromatography with other
separation methods such as HPLC and
electrophoresis facilitates protein separation. Recently, Hou et al. [40] have
developed a 96-well plate proteomic
reactor for gel-free processing of minute
amounts of complex proteomic samples.
The
device
performs
multiplexed
trapping, enrichment and biochemical
processing of proteins resulting in concentrated peptide solutions ready for
mass spectrometric analysis and this plate
proteomic reactor was coupled to size
Review

exclusion chromatography for large scale

identication of proteins.
The main advantage of SEC is that it
can be conducted under either denaturing
or non-denaturing conditions and it has
limited use in the study of protein separation using the tiny dierences in the
molecular size between modied and
unmodied proteins.

Capillary Chromatography
of Peptides
The small sample volume and low
abundance of proteins and peptides
present in biological uids and protein
digests are often a limiting factor for
analyses with conventional HPLC columns of 4.04.6 mm I.D. Using columns
with smaller inner diameters and shorter
length demands the utilization of fully
optimized equipment. The elution volumes of peptide analytes with l-HPLC
systems (when gradient elution conditions are employed) are considerably
smaller compared to conventional columns thereby permitting higher mass
sensitivity in diode array UV (DAD) or
laser induced uorescence (LIF) detection to be achieved.
In l-HPLC the peptide analytes are
transported through the column by a
hydraulic ow, with selectivity achieved
through the interaction of the peptide
with the stationary phase. In the case of
RP-HPLC selective separation of peptides on bonded silica sorbents is mainly
due to an adsorption/desorption mechanism and relies only to a limited extent on
partition eects [4146]. Fundamental
studies of capillary chromatography have
been performed by Knox and Grant [47]
and Smith and Escens [48] providing a
theoretical and practical framework for
the application capillary chromatography
using reversed and mixed mode sorbents.
Recently monolithic capillary columns
with surface-immobilized mannan have
been introduced for afnity-based micro
column separations by nano-liquid chromatography and capillary electrochromatography [49].

Monolithic Columns
Monolithic columns are attractive for the
purication of large biomolecules like
peptides because of their good ow
properties. Monoliths based on three
Chromatographia 2007, 65, May (No. 9/10)

dierent chemistries have been modied

into metal anity columns. Many reports
regarding application of monolithic columns for separation of peptides have
been discussed by Svec et al. [50]. Recently Peterka et al. [51] reported that
convective interaction media metal-chelate monoliths exhibit ow-unaffected
resolution and dynamic binding capacity
in the range of 1830 mg mL-1 for pure
and crude samples. These results indicated that the monoliths could be used
for purication of histidine-containing
proteins providing high productivity on
an analytical as well as on an industrial
level. Bedair M and Rassi ZE [52] reported that monolithic capillary columns
with immobilized manner are viable stationary phases for performing the selective isolation and concentration of
mannose/mannan binding proteins in dilute samples by afnity nano-LC and
afnity capillary electrochromatography
nanoproteomics. Severine et al. [53] described the preparation of a monolithic
phase, based on lauryl methacrylate as
functional manomer and a cyclohexanol,
ethylene glycol mixture as porogen in a
capillary format for chromatoghraphic
purposes. This column was successfully
used for separation of a cytochrome c
digest sample. Xiong et al. [54] described
the potential of silica monolithic columns
in peptide separation for proteomics.
Acrylate-based porous polymer monoliths have been used for separation of
peptides by reversed-phase electrochromatography by Shediac et al. [55]. Use of
monoliths to their full extent is limited by
nano LC instrumentation. In addition,
nano electrospray interfaces function best
at ow rates below 1lL min-1 [56]; to
overcome this to some extent Rievx et al.
[57] developed a 50 lM reversed phase
silica monolith in a nano LCMS set up.
Further reversed-phase monoliths investigated in this study appear to offer viable
alternatives to the recently introduced
UPLC systems.

Reversed-Phase High
Performance Liquid
Chromatography
Reversed-phase chromatography can be
used as the main separation procedure
for moderately complex peptide mixtures
prior to tandem mass spectrometric
analysis, but it is generally considered to
have insucient resolution for the anal-

513

ysis of more complex mixtures. While the

mass spectrometer can perform mass
measurements on several co-eluting peptides, if many peptides co-elute then the
MS instrument cannot fragment them all
before they nish eluting from the column, and therefore valuable information
is irretrievably lost. One interesting approach that has been used to circumvent
this problem is the iterative analysis of
aliquots of the same sample, using very
narrow mass ranges for the initial peptide
mass measurements of an MSMS scanning regime [58]. This technique therefore
uses the mass selection of peptides in the
mass spectrometer as an extra dimension of separation in the analysis of a
complex mixture. This approach is necessarily limited in application to those
cases where large amounts of sample are
available for analysis. It has been used for
example in LCMSMS analysis of the
proteome of normal human urine, in a
study in which 124 different gene products were identied [59]. In reversedphase HPLC the peptides are mainly retained due to hydrophobic interaction
with the bonded silica phase. Polar mobile phases, such as water mixed with
methanol or acetonitrile, are used to elute
the peptides which are eluted in order of
decreasing polarity (increasing hydrophobicity). The most popular reversedphase packing is C18 in which octadecasilyl groups are bonded to the silica surface [60, 61].

One-Dimensional
and Two-Dimensional
Liquid Chromatography
For one-dimensional liquid chromatography separation (1D-LC) reversed-phase
liquid chromatography is used in most
cases. The separation mechanism of RP
HPLC is a hydrophobic interaction with
the column material coated with alkyl
chains (e.g. C4, C8 or C18). The elution is
usually performed under acidic conditions by a gradient of water plus a watermiscible organic solvent like acetonitrile.
To enchance separation prior to MS
analysis, usually two (2D-LC) or even
multidimentional liquid chromatography
(MD-LC) chromatographic steps based
on dierent separation mechanisms, (so
called orthogonal separation mechanisms) have to be combined. Twodimensional LC requires less sample
handling and can be more easily auto-

514

mated. In addition, it can be coupled to

mass spectrometry either on-line via
electrospray ionization (ESI) [6264] or
off-line after spotting onto a target plate
for matrix-assisted laser desorption ionization (MALDI) [65, 66]. One-dimensional separations of complex samples are
still popular due to less demanding
instrumentation and easier operation
[67]. In addition, Davis [68] has shown
that, when the ratio of the number of
components relative to the peak capacity
is larger than about 0.5, many more
peaks (singlets, doublets, etc) will be observed in 1D separations compared to 2D
separations given the same peak capacity
for both. Therefore optimizing the peak
capacity in a 1D separation remains of
great importance.

The Role of Liquid

ChromatographyMass
Spectrometry in Proteomics
Chromatographic approaches to the
separation of proteomes are being widely
pursued as an alternative to gel-electrophoresis. Currently most chromatographic separations in proteomics are
done in RP-LC mode, because of its
compatibility with MS [69]. A variety of
multidimensional chromatographic approaches to separating proteins and
peptides have been described [7075].
Mass spectrometers are the workhorses of proteomics [7681] they are
used to measure the mass to charge ratios
(m/z) of biological molecules. In turn, the
m/z values can be used to calculate
molecular weights as well as determine
primary sequence information in tandem
mass spectrometry (MSMS) experiments. The goal of accurate mass measurement is to obtain an experimental
value for a biological molecules molecular weight with as little error as possible
[8286]. Generally, instruments that are
capable of higher resolution will also be
able to produce more accurate mass
measurements. Mass resolution may
determine by using the following equation.
Resolution m=zDm=zFWHM
Where m/z represents the centroid mass
to charge ratio of the ion signal and
Dm/zFWHM is the peak width at half
maximum. Mass measurement error is
reported as either a percentage of the
theoretical m/z value or in parts per mil-

Chromatographia 2007, 65, May (No. 9/10)

lion depending on how closely the value

can be measured as follows:
Smaller mass measurement error gives
higher condence in the identication of
the protein or peptide.
Four primary components make up a
mass spectrometer, the vacuum system,
ion source, mass analyzer and detector.
The purpose of ion source is to ionize the
analyte molecules and isolate them in the
gas phase, so that their m/z values can be
measured. The type of ion source used
determines the types of ions. The types of
ions that are formed in the mass spectrometry experiment, two ionization
methods, matrix-assisted laser desorption
ionization (MALDI) and electrospray
ionization (ESI) are mainly used in the
analysis of peptides and proteins by mass
spectrometry [87]. With the advent of soft
ionization techniques such as ESI and
MALDI coupled with various mass analyzers including the ion-trap, time of ight
(TOF), quadrupole and Fourier transform
ion cyclotron (FTMS) the analysis of
large intact proteins is now possible [88].
In MALDI, a solution containing the
analyte of interest is mixed with another
solution containing the matrix and
deposited on a sample probe. When dry
the analytes co-crystallize with the matrix. Desorbing the deposit with a laser
ionizes the analytemolecules. This method produces ions that correspond to the
protonated molecules of the analytes.
Tanaka and coworkers rst discovered matrix assisted laser desorption
ionization using ne metal particles
(
5 lm in size as the matrix [89]. Karas
et al. [90] rened this method by changing
the matrices, using organic acids, usually
cinnamic or benzoic acid derivatives, instead of metal particles. This modication greatly increases the versatility of
MALDI MS so that proteins, lipids,
carbohydrates and nucleic acids can be
analyzed. Sample preparation conditions
(e.g. solvent, matrix, and additive choices) must be optimized in each case.
However MALDI technique suers
certain inherent disadvantages, such as
MALDI exhibits low sensitivity for
proteins above about 30 kDa and the
mass spectrum is often obscured by
matrix-related ions.
Proteins
undergo
fragmentation,
resulting in the generation of broad
peaks and loss in sensitivity, which restricts MALDI to analysis of peptides
only.

Review

LCMS coupled with ESI and other

interfaces is widely used for the direct
detection and identication of proteins
[9193]. The molecular structure of the
main products can be sensitively analyzed
by MS with prior separation by liquid
chromatography, enabling the detection
of characteristic binding formations. In
tandem MS (LCMSMS) the ion of
interest is selected with the rst analyzer
(MS-1), collided with inert gas atoms in a
collision cell, and the fragments generated by the collision are separated by a
second analyzer (MS-2).
In ESIMS a solution containing the
analyte of interest is sprayed from a capillary jet by using a high voltage forming
small droplets in the gas phase [94]. These
droplets evaporate, leaving multiply
charged ions; in proteins and peptides the
ions are multiply protonated or deprotonated. The rst electrospray ion source
was reported by Yamashita and Fenn
[95]. The development of nano-electrospray MS by Wilm and Mann [96] has
extended this method to signicantly
smaller volumes and thus much smaller
amounts of analyte. However this method
suffers from some drawbacks, such as
Only those molecules which are suciently polar to allow attachment of a
charge can be analysed.
Samples have to be substantially free of
salts and detergent and need purication or concentration by liquid chromatography or use of a reversed phase
infusion mode.
Sensitivity is poor for large molecules
as signal is distributed over many
charge states and the complexity of a
protein similarly splits the signal into
many peaks.

Fourier Transform Ion

Cyclotron Resonance
Spectrometry (FTICR-M)
FTICRMS is the MS technique of
choice for combination with capillary
isoelectric focusing (CIEF) and ESI
[97102] because of its high resolution
and high sensitivity for peptide analysis.
On the ion cyclotron resonance (ICR)
principle, the ions produced by the various ionization methods, including electro
spray, are stored in an homogenous
magnetic eld where they move in circular orbits with a frequency (cyclotron

Review

frequency) determined by the product of

the magnetic eld strength and the mass
to charge ratio of the ions [98]. This
magnetic eld strength force can be expressed as

Fs qv  M
Where Fs is the force, q is the charge of
the ion, v is the velocity of ion and Mmagnetic strength. Two competing forces
can explain the cyclotron motion, in
which outward directed centrifugal force
Fc is compensated by the magnetic eld
strength force Fs. At stable transition the
forces are equal, so we may describe the
equation for this principle as follows.
Fs = Fc

qvM m

v2
v
M
) q :
r
m
r

Where v/r is the cyclotron frequency

velocity of the ion

radius of the transation
= cyclotron frequency

above equation evaluates the results in

radiation per second and has to be converted to frequency in Hertz by dividing
with 2p.
The advantage of (CIEF) coupled online with ESIFTICRMS have been
fully demonstrated by the analysis of cell
lysate harvested from E.coli cultured in
normal and isotopically depleted media
[97]. This method enabled the detection
of about 900 proteins with unique putative protein masses plotted as scan number versus molecular mass to obtain a 2D
display. Recently Song et al. [103] described a novel design to economically
convert a capillary liquid chromatography system with a capillary binary pump
in to an easy to use nano-LC system for
proteomic study.
CIEFESIFTICRMS analysis has
been further extended to capillary liquid
chromatographyESIFTICR MS [104
107]. The use of tandem FTICRMS
seems to be particularly promising for
high-throughput peptide identication,
because partial sequence information can
be obtained [108]. Capillary LCFTICR
analysis of the tryptic peptides of a radio
durans cell lysate provided a 2D map
consisting of over 13,000 peptides
spots which were drawn based on their
molecular mass and LC elution time, in a
single run [109]. Recently Jyan et al. [110]
reported proteomic proling of human

Chromatographia 2007, 65, May (No. 9/10)

urinary proteome using nano-HPLC

coupled to ESIMS. The experimental
results demonstrated that a total of 2,283
peptides, corresponding to 311 unique
proteins were identied from a human
urine sample, in which 104 proteins were
at a high condence level.

Orbitrap Mass Analyzer

The Orbitrap as a novel mass analyzer
has drawn attention recently due to its
high analytical performance and relatively small size [111114]. This mass
analyzer is an electrostatic trap wherein
tangentially injected ions rotate around a
central electrode, being conned by
applying an appropriate voltage between
the outer and central electrodes. Mass
analysis is based on image current detection of frequencies of axial oscillations
[115]. It provides high mass resolution
(up to 150,000), large space charge
capacity, high mass accuracy (15 ppm),
a mass/charge range of at least 6,000 and
a linear dynamic range greater than 1,000
[116]. Hu et al. [112] have reviewed the
use of this analyzer. Recently Peterman
et al. [114] have reported metabolite
identication of the anti-depressant nafazone in human liver microsomes with
this analyzer.

Time of Flight MS (TOF)

The ionized analyte molecules are accelerated by a xed amount of kinetic energy and travel down a ight tube and
because ions of dierent mass have different velocities they reach the detector at
the far end of the ight tube at dierent
times to give a spectrum [115]. The height
of each peak is proportional to the
number of ions of that particular mass to
charge ratio. The TOF equation derived
from equating the kinetic energy of an
ion with the potential energy of an ion in
an electric eld is given in the following
equation.
2
m= zvTOF 
z
L2

Where v is the accelerating voltage. TOF

is the ion time of ight, and L is the
length of ight tube.
TOF instruments were popular in the
early days of MS but has severe discrimination problems that modern

515

instruments have overcome. However,

TOFMS requires a pulse of ions (that is
a well-dened start time). It was not
readily compatible with ionization methods available so the following additional
analyzers have been used.
Orthogonal TOF (oTOF) [116] and
hybrid quadrupole (CP-TOF) [117122]
mass analyzers can be interfaced with
both MALDI and ESI sources. The
advantage of oTOF over axial TOF is
that the ion source is decoupled from the
mass measurement and because of this
external calibrations are much more
accurate in oTOF. The most recent
development in TOF mass spectrometry
is the MALDI-TOF-TOF mass spectrometer [123127]. In these instruments
an axial linear TOF is used to separate
the ions for mass selection, a collision cell
is placed after the mass selector for
fragment ion production, and then the
fragment ions are reaccelerated in a second source for mass measurement using
an axial reected TOF. These instruments
are very powerful accurate mass measurement of peptides and their fragment
ions. They also have a very fast response
time.

Quadrupole MS
Mass separation in a quadrupole instrument is a result of ion motion in a dynamic radio frequency electric eld and is
dependent directly on the m/z of the ion
[128]. Mass analysis is a function of rf
voltage and direct current (dc) voltages
applied to the four quadrupole rods. By
altering the relative contributions of the
dc and rf components the width of the
band pass area and therefore the resolution can be adjusted.
Quadrupole instruments act as a mass
lter allowing only a certain mass to pass
through and in process scanning of
amplitude of the electric eld and
recording on an ion detector gives the
mass spectrum. Depending on the physical parameters of the quadrupole, the
upper m/z limit can vary from 300 to
4,000, the mass accuracy is generally in
the hundreds of ppm. The mass resolution is a function of the ratio of the rf and
dc voltages and is often varied such that
unit resolution can be obtained over the
whole mass range. For MSMS analysis,
three quadrupoles can be congured together (to form a triple quad). The rst
and third are used for scanning whilst the

516

middle quadrupole is used as a collision

cell. Ions in the second quadrupole are
fragmented by collision-activated dissociation (CAD): low energy collisions with
a back ground gas such as nitrogen.
The radio frequency of the analyte can be
calculated by the following equation
[129].

V t Vdc Vrf cos x t

V(t) = voltage at time t
Vrfcosxt = radio frequency of the component
Vdc = direct potential

using LCFTICRMS peptide separation

to give an estimated peak capacity of
>6 107. Poppe [136] showed that
smaller particles make faster separations
without sacricing efciency. However,
the cost of using smaller particles is a
dramatic increase in pressure drop, to
circumvent this limitation ultra highpressure liquid chromatography [137,
138] can be used with smaller particles
(1.02.0 lm) to produce high plate
counts in isocratic chromatography and
large peak capacities in gradient chromatography [139].

Peptide Database
The quadrupole mass spectrometer
has become an important type of MS and
still excels relative to the other instruments in terms of quantication.

Peak Capacity
A large peak capacity is required for the
separation of the complex mixtures involved in proteomics peak capacity or the
number of signals that can reside in an
area of two dimensional space [130], is the
most common parameter used to evaluate
the applicability of a separation method
to the analysis of complex mixtures [131,
132]. Peak capacity of the separation was
increased by the way of decreasing the
overlap as indicated by Davis and
Giddings [133]. Recently Xiaoli et al. [134]
showed that the highest peak capacities
can be obtained with the column format
that provides the highest isocratic efciency. Their work aimed to maximize the
peak capacity using long columns packed
with larger diameter particles and these
particles should preferably be pellicular.
Another way of improving peak capacity
is to use higher temperatures (e.g.
>100C) where increase in the diffusivity
of the solute reduces the peak width. Peak
capacity can be calculated from the peak
width measured at 13.4% of peak height
at the gradient (separation) time tg by
using the following equation

p 1

tg
:
d

The high degree of orthogonality between LC and MS signicantly enhances

applications to very complex peptide
mixtures because LCMS disperses
signals over a large 2D area. This was
clearly demonstrated by Shen et al. [135]
Chromatographia 2007, 65, May (No. 9/10)

Computer-assisted methods to handle

experimental data essential in proteomics. The computer-assisted interface
discipline deals with accessorize storage,
management, access and processing of
peptide separation data. This discipline
assists researchers in dierent areas
resistant to simplistic analysis, including
inference of complex gene interaction
networks and cascades of intermolecular
interactions in development behavior of
cell ensembles in the brain and immune
system. Modeling will indispensable for
better understanding of complex human
diseases, including cancer and AIDS.
Common objectives of the majority of
the databases are maximization of annotation, minimization of redundancy and
integration or at least linkage to other
databases, currently the number of proteomics databases available in the World
Wide Web is increasing [140142]. Some
of these are as follows- database of
interacting protein (http://www.dip-doembi.ucla.edu), proteinprotein interaction
database (http://www.ebi.ac.uk/intact),
human protein reference database (http://
www.hprd.urdl), and human protein
interaction database (http://www.hpid.
org/).

Conclusions
and Perspectives
In this review we have focused on several
recent applications of chromatographic
technologies and in particular liquid
chromatography coupled to mass spectrometry in the eld of proteomics.
However, improved methods will be
required to advance understanding of
cellular function using proteomics
Review

techniques. The objective of chromatography in proteomics is to provide high

resolution, high-speed, high sensitivity
and highly specic separation of extremely complex mixtures and to facilitate mass spectrometric detection and
quantitative measurement. Technologies
exhibiting promise include novel monolithic stationary phases in capillary columns,
high-temperature
separation,
capillary electrochromatography and immuno metal anity chromatography
coupled to suitable mass detectors.

Acknowledgments
The author (Srinubabu Gedela) express
hearty thanks to Professor A. Ravi
Prasad (Department Head), Professor
Allam Appa Rao (Principal AU Engg.
College) to nominate IYBA 2006;
Professor V.L.N. Seshgiri Rao (Pharmaceutical Analysis), Dr. B.V.V. Ratnam (Johns Hopkins University, USA)
for their Moral support, Lotus labs
(Actavis), Bangalore where I enhance
my knowledge on biological samples
handling and nancial support from
TEQIP grants.

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