Biochimica et Biophysica Acta 1646 (2003) 1 – 10

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Review
Large-scale protein identification using mass spectrometry
Dayin Lin a, David L. Tabb b, John R. Yates III a,*
a
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA
b
Department of Genome Sciences, University of Washington, Box 357730 Seattle, WA 98195, USA

Received 2 September 2002; received in revised form 25 November 2002; accepted 3 December 2002

Abstract

Recent achievements in genomics have created an infrastructure of biological information. The enormous success of genomics promptly
induced a subsequent explosion in proteomics technology, the emerging science for systematic study of proteins in complexes, organelles,
and cells. Proteomics is developing powerful technologies to identify proteins, to map proteomes in cells, to quantify the differential
expression of proteins under different states, and to study aspects of protein – protein interaction. The dynamic nature of protein expression,
protein interactions, and protein modifications requires measurement as a function of time and cellular state. These types of studies require
many measurements and thus high throughput protein identification is essential. This review will discuss aspects of mass spectrometry with
emphasis on methods and applications for large-scale protein identification, a fundamental tool for proteomics.
D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Protein identification; Proteomic; Large-scale; Mass spectrometry

1. Introduction and presumably needs more genes to do so. A recent

The last decade in science belongs to genome sequenc-
ing. Besides the human genome sequencing project [1,2],
many other scientifically, medically, and economically
interesting organisms were sequenced. As of April 2002,
the Entrez database of the National Center for Biotechnol-
ogy Information had a collection of completed or partially
completed genomes representing more than 800 different
organisms http://www.ncbi.nlm.nih.gov/. More recently,
two separate research groups reported high-quality draft
sequences of the rice genome [3,4], an agricultural research
milestone that will lead to advances in studies of this
important food grain that provides more calories than any
other single food in the world [5]. Interestingly, a greater
number of genes were predicted in the rice genome than was
found in the rough draft of the human genome. A less
complicated organism (fewer cell types, shorter life span)
such as rice might be expected to contain fewer genes, but
rice leads an immobile existence and therefore must respond
to a greater number of environmental and biotic challenges
* Corresponding author. Tel.: +1-858-784-8862;
fax: +1-858-784-8883.
E-mail address: jyates@scripps.edu (J.R. Yates).
interpretation of the fewer than expected human genes
suggests that this may result from the need for an advanced
immune system to fight off disease [6]. Distinguishing
between self and non-self may ultimately limit the number
of different gene products the immune system can tolerate
and still provide adequate protection [6]. The implications
are that the human genome has had to develop alternate
methods to create functional diversity and this may be
accomplished through covalent modification of proteins
and alternate splicing of genes. Thus, the challenges to
understand the human biological system will require sophis-
ticated protein biochemical methods. Most certainly pleio-
trophy will play a significant role in creating the necessary
functional diversity required to create the 250+ different cell
types found in humans. Fortunately, the rapid completion of
genome sequences has provided the essential information to
link genes to gene products—proteins, the building blocks
for cellular functions. A completed genome sequence pro-
vides the infrastructure for high throughput and large-scale
protein analysis and these techniques will be essential to
establish the functional framework of all proteins of an
organism.
New technologies, based on the use of genomic informa-
tion, are accelerating the pace of biological discovery. Protein
biochemistry has been a beneficiary of these advances which

1570-9639/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1570-9639(02)00546-0
2 D. Lin et al. / Biochimica et Biophysica Acta 1646 (2003) 1–10
have come about through synergies of genomic data and
advanced mass spectrometers. Current high-sensitivity meth-
ods using mass spectrometry for protein identification have
vastly lowered detection limits [7], permitting the facile
analysis of proteins with low cell copy numbers from fewer
numbers of cells [8]. An increasing level of automation is
allowing the development of high-speed proteomic experi-
ments. In addition, the quality of bioinformatics continues to
improve; making predictions of gene products deduced from
genome information increasingly accurate. With this data-
base infrastructure in place, algorithms designed to match
mass spectrometry data to known protein sequences have
become possible [9,10]. Isolated proteins can be matched to
protein sequences by peptide mass fingerprinting, while more
complex mixtures of proteins can be analyzed through
tandem mass spectrometry. These improvements in instru-
mentation and automation make possible experiments far
greater in scale than ever before.
Proteomics can be used to help define the functions and
interrelationships of proteins in an organism [11,12]. The
basic experiments to achieve this aim are commonly prac-
ticed in biochemistry and molecular biology. In the past, the
rate-limiting step has been the analytical process through
which the proteins observed in an experiment such as an
immunoprecipitation can be identified. To inventory a cell’s
complete protein content and dissect its protein interaction
network requires protein identification technologies that can
operate at extremely high throughput and sensitivity. Further
complicating the analysis of proteins are the chemical
modifications that may occur either co-translationally or
post-translationally to modify or regulate their functions.
Covalent modification events include phosphorylation,
methylation, glycosylation, prenylation, formylation, or
acetylation. Other modifications of proteins include proteo-
lytic cleavage, oxidation of some amino acids, or cross-
linking events. All processing adds greatly to the complex-
ity of protein analysis. Modifications may also change both
chemical and physical properties of individual proteins,
such as chromatographic behavior, mass, and ionization
efficiency. Although the complexity of the proteome is a
challenge for its analysis, techniques to handle structural
and modification features have evolved to make mass
spectrometry an important technology for large-scale protein
identification [9].
2. A paradigm shift: from sequencing to identification
Edman sequencing has been the gold standard for protein
sequencing for the last 25 years [13]. For unambiguous
sequencing, a protein is purified to homogeneity prior to
sequencing. The sample then undergoes cycles of Edman
degradation reactions to remove the N-terminal amino acid
which is collected at the end of each cycle for identification
by liquid chromatography [14]. Each cycle requires 30 – 60
min. Edman sequencing is complicated by a blocked N-
terminus and incomplete purification [15]. A recent inno-
vation in the use of Edman degradation has improved the
analysis of simple protein mixtures by deconvoluting the
mixed sequence from each cycle using sequences from
databases [16]. Other traditional approaches for protein
identification include the use of antibodies to perform
Western blots [17]. Antibody use, however, is impaired by
non-specific binding and by the availability of antibodies to
all proteins [18]. As genome sequence information has
accumulated, the paradigm has shifted from sequencing to
identification. This situation has been facilitated by advan-
ces in ionization and mass analysis techniques for mass
spectrometry and the ability to correlate mass spectrometry
data of peptides and proteins to sequences in databases.
3. Ionization techniques for biomolecules in mass
spectrometry
Mass spectrometry analysis of peptides and proteins relies
exclusively on soft ionization techniques that create intact
gas-phase ions from biomolecules. The creation of intact
molecular ions enables accurate measurement of molecular
weight. The electrospray ionization (ESI) and matrix-assis-
ted laser desorption/ionization (MALDI) techniques were
developed more than 10 years ago and revolutionized the
analysis of biomolecules [19 – 22]. Biomolecules are often
polar and charged, thus conversion of solution or solid-phase
ions to gas-phase ions is not an energetically favorable
process. ESI and MALDI methods are currently the principal
methods for peptide/protein ionization and they have been
linked to high-throughput sample preparation techniques
[23].
3.1. Electrospray ionization
ESI operates at atmospheric pressure and produces tiny
charged solvent droplets when a high electric potential (2– 5
kV) is set between a capillary and the inlet to a mass
spectrometer. By using a drying gas or heat in the atmos-
pheric pressure interface, the charged droplets shrink and
eventually desolvated ions are desorbed from the droplet
[24]. The ESI process can be used to produce negative or
positive ions, but typically peptides or proteins are analyzed
as positive ions using the capillary as an anode and the MS
inlet as the cathode. ESI uses a steady stream of solvent to
produce a continuous beam of ions and thus is readily
coupled to HPLC. When operating at high solvent flow
rates of 100 Al/min to 1 ml/min, a nebulization gas (or
sheath gas) is needed to assist the solvent dispersion. At low
flow rates, electrospray can be induced by electric potential
alone. Two operating regimes have been defined at low flow
rates. The first is microelectrospray, where the flow rate is
approximately 100 –500 nl/min; and the second is nano-
electrospray, where a flow rate of 100 nl/min or less is used
[25,26]. These low flow rates create smaller droplets and a
 D. Lin et al. / Biochimica et Biophysica Acta 1646 (2003) 1–10
much reduced Taylor cone, allowing the bulk of the spray to
be directed into the mass spectrometer’s inlet [26]. As a
result, much less sample is required than at higher flow rates
because of the concentration-sensitive nature of electro-
spray. Another characteristic of ESI is the production of
multiply charged ions which lowers the m/z values (over
singly charged species, z=1) for higher molecular weight
compounds and thus allows measurement of m/z values on
mass spectrometers with limited m/z ranges. Furthermore,
multiple protonation of peptides and proteins promotes
more facile amide bond fragmentation when the ions are
activated for dissociation. These features of ESI have led to
its widespread adoption for proteomics research.
3.2. Matrix-assisted laser desorption/ionization
MALDI uses energy from lasers rather than electrical
potential to ionize biomolecules. MALDI uses small UV
absorbing molecules to co-crystallize with peptides/proteins
on a sample plate [22]. Ionization occurs when these matrix
molecules absorb the energy provided by a laser (usually 337
nm). Release of the energy causes a rapid thermal expansion
of matrix and analyte into the gas phase. Proton transfer from
analyte to matrix may result in charge reduction to the singly
charged ion observed in the gas-phase [27]. The most
commonly used matrix molecules are a-cyano-4-hydroxyci-
naminic acid for peptides and polypeptides less than 5000
Da, and 3,5-dimethoxy-4-hydroxy-cinnamic acid, or sina-
pinic acid, for proteins. MALDI produces predominately
singly charged ions and is less sensitive to salts in the buffer
than ESI, although salt and matrix adducts of analyte ions
can form. Because MALDI depends on pulsed laser radia-
tion, ions are created in bunches or packets, consequently,
mass analyzers capable of analyzing ions created in an
intermittent fashion are required. The most common mass
analyzers used with MALDI are time-of-flight (TOF) mass
spectrometers with growing interest in using this ionization
technique with ion trap mass spectrometers (described
below). A recent innovation in MALDI sources is the
operation of this device at atmospheric pressure [28]. This
method of operation simplifies source design and allows the
use of MALDI with sources originally designed for ESI.
Thus, the two different ionization methods can be used
interchangeably with the same instrument.
4. Mass analyzers for proteomic mass spectrometry
As ions exit the ion source, they pass into a mass analyzer.
The mass analyzer is responsible for separating ions by their
mass-to-charge (m/z) ratios. Mass analyzers use electric and/
or magnetic fields to manipulate ions in a mass-dependent
manner. Quadrupole (Q) mass analyzers use a radiofre-
quency (RF) voltage applied to four metal rods with RF
voltage of alternate polarity placed on opposite rods. A direct
current (DC) voltage is overlayed on the rods. The ratio of
3
RF to DC voltage selectively stabilizes the trajectory of ions
of particular m/z value as they pass through the analyzer. Ion
current is recorded at a detector as ions exit the analyzer [29].
Quadrupole ion trap (IT) analyzers create three-dimensional
RF fields to trap ions in the center of a ring electrode [29,30].
The field can be manipulated to selectively eject ions of a
particular m/z value to a detector to record the m/z ratios or to
selectively retain a particular m/z value for collision-acti-
vated dissociation (CAD). CAD can be used to fragment ions
by exciting the trapped ions to increase their motion causing
hundreds to thousands of ion-molecular collisions with the
helium bath gas. The resulting fragment ions are then
sequentially ejected to the detector [30]. TOF mass analyzers
accelerate a packet of ions with a set of electric potentials and
differentiate them by the time they take to traverse a flight
tube [31]. An m/z value can be calculated from the time
required to move from the ion source to the detector. Mag-
netic fields can also be used to measure m/z values by
trapping ions in a static magnetic field (Ion Cyclotron Mass
Spectrometry also called Fourier Transform Mass Spectrom-
etry, FTMS). Ions rotate around the magnetic field with a
cyclotron frequency related to their m/z value. By perturbing
ion motion in the cell, the ion motion goes into coherence
and an electric signal can be measured between two detector
plates (detector plates detect an ion current as the packet of
ions moves from one side of the cell to the other). As the
motion of the ions decays back to their natural cyclotron
frequencies, the signal deteriorates. By recording the signal
decay, frequencies can be calculated using a Fourier trans-
form to calculate very accurate m/z values [32]. Hybrid mass
spectrometers that combine different types of mass analyzers
have been constructed to produce unique capabilities. For
example, quadrupole-TOFs are instruments that combine a
quadrupole mass analyzer for ion selection in a tandem mass
spectrometer mode and TOF analyzers to record m/z values
with high resolution [33]. Ions are dissociated in a collision
cell between the two mass analyzers. More recently, TOF
mass analyzers have been combined to create tandem mass
spectrometers (TOF– TOF). In this instrument, m/z values
are selected by their TOF and all others are deflected from
the flight path. The ions then pass into a collision cell and
undergo high-energy collisions with an inert gas such as
helium, causing fragmentation of the ions. This prompt
fragmentation of ions is referred to as collision-induced dis-
sociation (CID). All of the above mass spectrometers are
effective for proteomic studies and differentiate on the basis
of mass range, mass accuracy, sensitivity, resolution and
cost. Ion trap and TOF mass analyzers are compatible with
MALDI because they trap packets of ions or analyze them
simultaneously, respectively.
5. Overview of protein identification by mass analysis
Complete protein identification involves correlating data
from a protein with its gene sequence and identifying the
4 D. Lin et al. / Biochimica et Biophysica Acta 1646 (2003) 1–10

site and type of modifications that may be on the protein
(Fig. 1). To unambiguously correlate an isolated protein’s
sequence to the sequence predicted through genome
sequencing, data indicative of amino acid sequence must
be derived from the protein. During the last several years,
two different approaches for MS-based protein identification
from complex mixtures have been maturing: ‘‘top-down’’
and ‘‘bottom-up’’ [34].
5.1. Top-down approach
The ‘‘top-down’’ strategy starts with an intact protein and
cleaves the protein in the gas phase rather than in solution.
The protein is fragmented inside the mass spectrometer to
create a ladder of ions indicative of the sequence. The
difference in m/z values of fragment ions define the position
and sequence of the amino acids in the protein. Several
methods and mass spectrometers are used for ‘‘top-down’’
analysis. ESI-FTMS is able to analyze intact proteins
because of its capability for high resolution. Proteins are
fragmented in this device using electron capture dissociation
(ECD) [35]. Other methods of ion dissociation have been
used in FTMS instruments, but ECD has the potential to be
a general method for protein fragmentation [36]. FTMS can
isolate individual m/z values (a specific charge state of a
protein, for example) and thus simple protein mixtures can
be introduced into the mass spectrometer and then frag-
mented separately. Proteins need not be purified to homo-
geneity and thus have less dependence on chromatographic
separation prior to MS analysis [37]. The high mass accu-
racy of FTMS instruments can detect protein sequence
errors and protein post-translational modifications [38 –
41]. Even as ECD improves, protein fragmentation is still
not as general as peptide fragmentation. Computer algo-
rithms to use the data derived from ‘‘top-down’’ methods to
identify protein sequences from databases have been devel-
oped [37,42]. However, the observed molecular weight of a
protein may differ from the predicted molecular weight
because of sequence errors, post-translational modifications,
or proteolyic processing. The use of protein fragmentation
techniques such as ECD has facilitated protein identification
using intact proteins and databases [43]. After a protein has
been identified, an accurate molecular weight can be enor-
mously useful in pinpointing the extent of modification of
the protein. This technique will have a significant role in the
identification of small proteins (<10 kDa) because they are
difficult to predict through bioinformatics and there is much
to be learned about their biological roles.
5.2. Bottom-up approach
The ‘‘bottom-up’’ approach identifies proteins by tandem
mass spectrometry analysis of peptides derived from diges-
tion of mixtures of intact proteins [44,45]. The resulting
peptide mixture is chromatographically fractionated and
introduced into a tandem mass spectrometer. Tandem mass
spectrometers can select individual peptide ions for analysis
from mixtures of ions. The fragmentation pattern derived for
each peptide is indicative of the amino acid sequence of the
peptide. This pattern can be used to search sequence data-
bases without first interpreting the sequence from the spec-
trum. Amino acid sequences are selected for further analysis
from the database on the basis of the molecular weight of the
peptide. The predicted fragmentation pattern for each peptide

Fig. 1. Schematic showing the ‘‘top-down’’ and bottom-up approaches to protein analysis and identification. In the ‘‘top-down’’ approach, the intact protein is
fragmented in the gas-phase to create sequence-specific fragment ions to aid in sequence analysis. The bottom-up approach uses proteolytic enzymes to create
peptides for sequence analysis usually by using multidimensional liquid chromagoraphy in combination with tandem mass spectrometry.
 D. Lin et al. / Biochimica et Biophysica Acta 1646 (2003) 1–10
Table 1
Publicly available algorithms for protein identification from mass
spectrometry data
Peptide mass mapping database matching tools
Mascot http://www.matrixscience.com
Mowse http://www.hgmp.mrc.ac.uk/Bioinformatics/Webapp/mowse
MS-Fit http://www.prospector.ucsf.edu
Pepsea http://www.pepsea.protana.com
ProFound http://www.proteometrics.com
Tandem mass spectrum database matching tools
Mascot http://www.matrixscience.com
MS-Tag http://www.prospector.ucsf.edu
Pepsea http://www.pepsea.protana.com
SEQUEST http://www.fields.scripps.edu/sequest
Sonar http://www.proteometrics.com
is then compared to that of the spectrum. When a prepon-
derance of the fragment ions match, it is considered a good
fit. Several algorithms have been developed to automate this
process for protein identification (see Table 1). Several
advantages exist in using tandem mass spectrometry data
for protein identification. For instance, there is a higher level
of certainty in the identification of proteins since the method
relies on several pieces of highly specific information. Also,
it is straightforward to identify the type and site of modifi-
cation [46]. Finally, proteins can be identified in mixtures
[44,47,48].
6. Large-scale separation methods in proteomics
Cells and tissues contain thousands of proteins spanning a
wide range of abundance. Mixtures of proteins or compo-
nents of cells and tissues can be fractionated by many
different methods including centrifugation, ion exchange
chromatography, size exclusion chromatography, affinity
chromatography, reversed-phase liquid chromatography,
and gel electrophoresis (GE). Multiple fractionation steps
are often required to simplify mixtures prior to analysis.
Ideally, a method that will separate a maximum number of
proteins in the fewest possible steps is desired. Two major
strategies for proteomic analysis, two-dimentional gel elec-
trophoresis (2DGE) and 2D-liquid cloromatography are
illustrated in Fig. 1.
6.1. Two-dimensional gel electrophoresis
2DGE is an important method for proteome analysis
because a large number of proteins can be separated in a
single experiment [49]. Proteins are separated in a first
dimension by charge using isoelectric focusing (IEF). The
charge-focused proteins are then separated in the second
dimension by size using SDS-polyacrylamide gel. Proteins
are then visualized by staining the gel with hydrophobic
dyes like Coomassie, silver ions, or fluorophores. Excess
reagent is washed from the polyacrylamide gel and the
stains adhere principally to the proteins, fixing their position
5
in the gel and allowing determination of pI and molecular
weight. The intensity of the stained protein also provides a
measure of how much protein is present and provides a
means for differential analysis. A new strategy for this type
of analysis involves the use of two fluorophores with
different emission wavelengths that allows premixing of
mixtures of proteins each labeled with the different fluo-
rophores [50]. After separation, the intensity of fluorescence
at each wavelength is determined, providing a relative
measure of how much of each protein is present in each
of the samples. 2DGE is very effective at separating charge
and size isoforms of the same protein providing a measure
of how a protein may be modified. 2DGE provides separa-
tion and visualization of the protein mixture but does not
explicitly identify proteins (data from past experiments can
be used in theory, although care must be exercised). A
second analytical step must be employed to identify the
proteins where proteins are excised from the gel, subjected
to proteolytic digestion, and identified or sequenced. A
2DGE can produce a wealth of information from each
separation and is very effective for differential analyses.
The strength of 2DGE is that it can separate up to 10,000
proteins [51]. This separation involves the use of narrower
pH gradients (e.g. 5 – 6, 6 –7, rather than 4 –10) during the
isoelectric focusing stage, and the use of larger amounts of
proteins to increase the dynamic range of the separation.
After the second dimension separation, the gels are aligned
to observe the whole separation. However, several draw-
backs can limit its effectiveness. Excising proteins from a
gel manually is time-consuming, and when hundreds or
more spots need to be processed from a single gel, automa-
tion becomes necessary. Robotic systems are available to
minimize the amount of labor required to perform these
steps. Many of the drawbacks of 2DGE stem from the
physical limitations of the separation. Most 2-D gels can
only focus proteins with a pI range between 4 and 10, thus
excluding very acidic and basic proteins. Proteins with
molecular weights below 15 kDa or above 200 kDa either
run off the gel or cannot move inside it unless special gels
are used, which generally require separate analyses to
resolve high molecular weight proteins and then again to
resolve small proteins. Buffers necessary for isoelectric
focusing limit the type of detergents that can be used,
limiting conditions to solubilize membrane proteins [52 –
54]. Integral membrane proteins do not appear on the gel
[55]. Low abundance proteins are often not observed on the
gel [56]. Several improvements to this technique, however,
have improved upon some of the issues [57,58]. Recent
efforts have focused on optimizing solubilization conditions
for membrane proteins and staining techniques. Mass spec-
trometry is now routinely used to identify proteins separated
by 2DGE.
Two different mass spectrometry techniques are com-
monly used to identify proteins separated by 2DGE. Be-
cause of the potential for high throughput analysis, MALDI-
TOF is the most attractive mass spectrometry method to
6 D. Lin et al. / Biochimica et Biophysica Acta 1646 (2003) 1–10
identify proteins from 2DGE [59]. Proteins are separated to
homogeneity, typically by 2DGE, and then trypsin or
another enzyme digests them into characteristic peptides.
A key component to mass mapping is cleavage of the
proteins into predictable peptide fragments, although less
specific proteases such as thermolysin have been used to
cleave peptides for mass mapping [60]. Mass spectrometry
(often MALDI-TOF) measures the m/z values of the pep-
tides, and an algorithm searches a protein database for
proteins that would produce peptides of these molecular
weights when cleaved with the same protease (see Table 1
for several programs used for this purpose). The quality of
the result depends on the purity of the sample, the accuracy
of the measurement, and the number of peptide masses
obtained. The peptide mass mapping method can be auto-
mated when coupled with robotic equipment that can deliver
processed samples from spots on 2-D gels. Mass mapping is
an attractive choice for proteomic studies involving 2DGE.
Shevchenko et al. [61] reported the identification of 150
yeast proteins by 2DGE using MALDI peptide mass map-
ping. In 1999, a 2-D yeast reference map was established,
and a combined 374 proteins were identified from several
different labs [62]. In an analysis of H. pylori proteome,
1863 spots were observed from multiple 2DGE separations
of prefractionated cellular material. Despite the observation
of so many proteins, only 126 of the proteins were identified
[63]. Single dimension gel electrophoresis can also be used
for large-scale protein identification studies. Two recent
analyses of immunoprecipitated yeast protein complexes
by using SDS-PAGE and mass spectrometry identified large
numbers of protein complexes [11,12].
6.2. Multidimensional protein identification technology
An alternate approach for the analysis of protein mixtures
uses a different paradigm to identify the proteins contained in
a mixture. This strategy uses the ability of tandem mass
Fig. 2. Preparation for a biphasic column for LC/LC/MS/MS analysis. A piece of fused silica capillary is pulled to have an opening of 5 Am. The packing of the
column is achieved by using a pressure bomb to force packing materials up to the column. RP and SCX materials are used sequentially to make a biphasic
column.
spectrometers to select and analyze peptides from protein
mixtures which have been proteolytically digested. The
peptides then serve as surrogate markers for the protein
sequence [44]. Proteins are identified by searching the
resulting tandem mass spectra through sequence databases.
Because digested protein mixtures create complex mixtures
of peptides, high-performance separation techniques are
required to resolve the peptides prior to entering the tandem
mass spectrometer [44,47,48]. This approach, when com-
bined with multidimensional liquid chromatography and
database searching, is called Multidimensional Protein Iden-
tification Technology, or MudPIT. This process fully auto-
mates the separation and identification of proteins from
complex mixtures [64,65]. The first step in any MudPIT
experiment is the enzymatic digestion of a protein mixture to
produce a more complex peptide mixture. A specially
packed biphasic liquid chromatography column (see Fig.
2) using a strong cation exchange (SCX) support as the initial
phase and reversed-phase (RP) material as the second phase
is used to separate peptides. The column is constructed from
fused silica and has an internal diameter of 100 Am and thus
eluent is delivered to the mass spectrometer at low flow rates
(100 – 200 nl/min). Peptides are stepped from the SCX phase
onto the RP phase in a series of salt steps that increase in
concentration. A subsequent RP gradient separates the pep-
tides and delivers the peptides into the mass spectrometer
after each salt step. Since each phase separates peptides by
orthogonal chemical properties, a high-resolution separation
among the peptides is achieved with the aim that the mass
spectrometer will analyze a more manageable number of
peptides at any point during the separation [66]. MudPIT
analysis of proteins from a cell can detect proteins over a
wide range of pI, abundance, and subcellular localization
[65]. The MudPIT method was used to identify proteins from
a yeast whole cell lysate by Washburn et al. [65]. From three
fractions (soluble proteins, peripheral membrane proteins,
integral membrane proteins) of the cell lysate, a total of 1484
 D. Lin et al. / Biochimica et Biophysica Acta 1646 (2003) 1–10 7

Table 2 described a program, DTASelect, to assemble, filters, and

Comparison between 2DGE and MudPIT methods
compare the results of database searches.
2DGE MudPIT
Pro . automation possible . unbiased analysis of all 6.3. Alternate multidimensional separations
proteins
. pre-cast gels available . less sample is needed
. good resolving power . highly resolving 2-D
The use of tandem mass spectrometry analysis of pep-
for proteins separation for peptides tides derived from digested protein mixtures has created an
. image data is quantitative . large number of proteins alternate approach for the analysis of experiments (see Table
can be detected 2 for comparison) [56]. Besides the direct on-line protocol
. widely used by researchers
of MudPIT, off-line multidimensional separation methods
Con . large quantity of sample . requires custom equipment
have been used to achieve the same basic aim as MudPIT
needed for column packing
. unsuitable for proteins . lack of direct quantitative [65]. Commercially available columns (ion exchange or
with extreme pIs or molecular information affinity) were used to fractionate peptides before each
weights or of low abundance . no commercially available fraction was subjected to LC/MS/MS analysis [69]. Gygi
. membrane proteins are hard system et al. [70] used two dimensions of off-line separation of
to detect
analyte before using LC/MS/MS. In this method, proteins
from S. cerevisiae were reduced and labeled at cysteines
with a biotin reagent followed by trypsin digestion [71].
proteins were identified. More significantly, low abundance Peptides are then separated by a strong cation-exchange
proteins, membrane proteins, small (less than 10 kDa) or step; each collected fraction is then passed through an avidin
large (greater than 180 kDa) proteins, and proteins with column to selectively isolate only biotin-tagged cysteine-
extreme pI values were all well represented in the data set. containing peptides, and finally, LC/MS/MS is performed
MudPIT appears to introduce very little bias in its sampling on fractions collected in the affinity step. This sequence of
of proteins from the cell but a key element is the sample separations is designed to increase the detection of low
preparation procedure (see Table 2). MudPIT type analyses abundance proteins. The use of an affinity step is meant to
require high throughput database searching techniques. reduce sample complexity, but the addition of multiple off-
Sadygov et al. [67] describe a multiprocessing database line steps requires the use of 5 –10 times the amount of
searching algorithm to increase the search speeds for the material as used in a MudPIT analysis. The drawbacks of
large number of spectra produced in a MudPIT experiment. off-line separations are decreased automation and increased
A second requirement is the need for computer algorithms to sample loss; this protocol requires milligram quantities of
assemble the data from the database search. Tabb et al. [68] sample in order to make up the sample loss between

Fig. 3. Schemes for large-scale protein identification methods. The gel-based 2DGE approach separates proteins from cell lysate by molecular weight and
isoelectric point. Each spot can then be excised and enzymatically digested for MS analysis. The liquid-based MudPIT method digests the proteins in the lysate
and separates peptides on a biphasic LC column for MS/MS analysis. Database searches are conducted for both approaches after MS data are collected.
8 D. Lin et al. / Biochimica et Biophysica Acta 1646 (2003) 1–10
chromatography steps. Conrads et al. [72] uses the high
mass accuracy of FTMS instruments to profile peptides
from digested protein mixtures. Measurement of m/z values
with high accuracy allows facile comparison between anal-
yses and provides in instances a mechanism to identify
proteins. To further ensure unambiguous identification of
the peptides, the complex peptide mixture is analyzed by
multiple LC/MS/MS experiments using ‘‘gas-phase’’ frac-
tionation on an ion trap mass spectrometer [73] (Fig. 3). By
reducing the scan range of the mass spectrometer (e.g. 500–
800), the number of ions available for data-dependent data
acquisition is reduced, allowing the acquisition of more
peptide ions then normally occurs during a single dimension
chromatographic separation. By repeating the process over
different mass ranges (e.g. 800 –1000, 1000– 1200, etc.)
more thorough coverage of the proteome is possible. In the
study by Lipton et al. [73] of D. radiodurans, an impressive
61% of the proteome was covered by analyzing cultures
grown under a variety of conditions.
A variation of the ‘‘bottom-up’’ approach that combines
features of the ‘‘top-down’’ approach is employed by Chong
et al. [74]. In this approach, proteins are separated by
multidimensional liquid chromatography and fractions col-
lected for digestion. Liquid isoelectric focusing has been
used together with reversed-phase liquid chromatography to
effect high-resolution separations [75]. After digestion,
fractions are then analyzed by MALDI-TOF to identify
the proteins present. Alternatively, the molecular weights
of proteins can be measured directly in the mass spectrom-
eter to create three-dimensional maps of proteins based on
pI, hydrophobicity and molecular weight to compare cells of
different states [76].
7. Future emphases
As challenges in protein identification are steadily over-
come, the focus in proteomics is shifting to other important
areas. Three areas of particular emphasis are ‘‘de novo’’
peptide sequencing, post-translational modification identifi-
cation, and quantitation of peptides by mass spectrometry.
De novo peptide sequencing by mass spectrometry
differs substantially from the peptide identification process
as embodied in SEQUEST and other algorithms, which rely
on the presence of the target peptide sequence in a supplied
database. The de novo challenge is to infer a peptide
sequence directly from the spectrum without external hints
as to what the sequence may be. Several attempts have been
made but success has been limited by data quality (such as
mass accuracy and signal to noise) and hindered by the
complexity of accurately predicting fragment ion spectra
[77,78]. It is unlikely that de novo sequencing will ever be
as sensitive as database searching methods.
Post-translational modifications play an important role
in determining protein function, but identifying peptides
containing these modifications poses many challenges.
Modified peptides, such as those bearing the important
phosphorylation modification, may produce fragment ion
spectra of greater complexity than normal peptides, compli-
cating their interpretation. At the same time, augmented
forms of existing database matching algorithms typically
take far longer to run on spectra when modification possi-
bilities are taken into account. Progress has been made in
solving this problem, but much work remains to be done
[79 – 84].
An increasing emphasis in proteomics is the quantitation
of protein content rather than simple determination of
presence or absence [85]. Several methods exist to quanti-
tate proteins and all rely on labeling proteins with a mass
label to differentiate the same peptides from two states of
the cells for relative quantitation. The first method uses
metabolic labeling to incorporate 15N into peptides [86]. A
second method uses proteolytic digestion in the presence of
18
O water to incorporate a label [87]. The last method uses
covalent labeling to incorporate the mass label [71,88 – 90].
Covalent labeling is targeted at reactive amino acid residues
and generally the label contains deuterium atoms in place of
hydrogen atoms. Labels have been developed for amino and
sulfhydryl groups. These methods allow the measurement of
relative quantitative states. Recently, Stemmann et al. [91]
have developed a method for the measurement of absolute
quantities of peptides. Because the method requires the
addition of a known quantity of a peptide standard, it is
not suitable for proteome-wide measurements. Biology is
moving to a more quantitative description of cellular pro-
cesses and mass spectrometry is poised to play a large role,
but all of this relies on high throughput identification of
proteins.
Acknowledgements
The authors acknowledge support from NIH grant
R33CA8165-01, NIH grant RR11823-05, and National
Science Foundation Graduate Research Fellowship.
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