DDT: TARGETS Vol. 3, No. 2 (Suppl.
), 2004 mass spectrometry in proteomics supplement
Analysis of large-scale MS data
sets: the dramas and the delights
Keiryn L. Bennett, Jan C. Brønd, Dan B. Kristensen,
Alexandre V. Podtelejnikov and Jacek R. Wiśniewski
The biotechnology and pharmaceutical industries are faced
with the serious challenge of consolidating the enormous quan-
tities of data that have been generated from high-throughput
proteomic applications. The bottleneck of data validation and
placement of the information obtained into sound biological
context urgently needs to be addressed. Here, we review the
issues that arise when analysing large quantities of data gener-
ated by liquid chromatography mass spectrometry, offer po-
tential solutions for data management and predict the future
direction of large-scale data analysis by mass spectrometry.
Keiryn L. Bennett* ▼ In the age of high-throughput proteomics,
Jan C. Brønd ‘more is better’ became the catch cry of the biotech-
Dan B. Kristensen nology and pharmaceutical industry. Companies
Alexandre V.
have invested vast amounts of capital in state-of-
Podtelejnikov and
Jacek R. Wiśniewski† the-art mass spectrometers, robotics to prepare
MDS Denmark samples for analysis, and automated systems to
Staermosegaardsvej 6 generate large data sets. These investments have
Odense M, DK-5230 neither been cost-effective nor significantly
Denmark
aided the pharmaceutical industry in discovering
*e-mail: kbennett@
mdsdenmark.com
new targets. Mass spectrometry is a core technol-
†e-mail: jwisniewski@ ogy in proteomic research; however, large-scale
mdsdenmark.com). proteome characterization has led to serious
bottlenecks in data interpretation.
The situation is eloquently captured by a re-
cent quote from Scott Patterson, “our ability to
generate data now outstrips our ability to analyse
it” [1], and reflects the current status for indus-
try and academia alike.There is now an evolution
towards statistically sound, biologically relevant
data sets: ‘quality, not quantity’ is the reality.
Efforts are concentrated on producing focused
data sets aimed at resolving the complexity of
biological samples. This is being achieved by a
synergistic relationship among sample prepar-
ation, mass spectrometry (MS) analysis of the
1741-8372/04/$ – see front matter ©2004 Elsevier Ltd. All rights reserved. PII: S1741-8372(04)02412-0
| reviews
biologically relevant samples, and data analysis
and integration.
Priorities of large-scale proteomic
analyses
The predominant methods of large-scale data gen-
eration are liquid chromatography mass spec-
trometry (LC–MS) of complex protein mixtures
[2–5], and matrix-assisted laser desorption ion-
ization (MALDI) MS profiling of tissue sections
[6,7] and sera [8]. The current needs of the in-
dustry with respect to the information required
from large-scale proteomic analyses are the de-
tection of pharmaceutically relevant proteins.
These include proteins involved in signal trans-
duction and the effects of inhibitors or activators
on the proteins; kinases and phosphatases involved
in pathway modulation; and receptors and associ-
ated ligands involved in intra- and intercellular
signalling. Identification of proteins such as tran-
scription factors, cell-surface proteins, secreted
proteins and biomarkers is a high priority for the
generation of drug targets, diagnosis of disease
and assessment of drug toxicity. Within these
classes of proteins, the challenge is to identify
low-abundance proteins and to provide quantitive
information.
Low-abundance proteins
Owing to the inherent nature of cellular com-
plexity, the detection and identification of low-
abundance proteins by MS is extremely challeng-
ing. To increase the chance that a protein of low
copy number will be detected, it is necessary to
reduce the complexity of the sample. There are
several approaches whereby sample complexity
can be simplified. One method is to fractionate
tissue and cells into subcellular components,
such as plasma membrane [9–11], mitochondria
[12] nucleoli [13] and lipid rafts [14,15].A second
www.drugdiscoverytoday.com S43
reviews | mass spectrometry in proteomics supplement
approach is to isolate protein populations by affinity chromatog-
raphy. This can be achieved by utilizing the inherent properties
of the protein via posttranslational modifications (e.g. phos-
phoproteome [19] and the glycoproteome [19]). Alternatively,
a specific amino acid can be labelled with a tag and, subsequently,
the tagged peptides can be isolated by affinity purification
[11,20].
Peptide and protein quantitation
There is an increasing demand for comparative proteomics in
which populations of specific proteins are compared between
two different states: for example, healthy versus diseased tissue.
There are three main methods for quantitation by MS. The first
approach employs in vivo metabolic labelling. Different cell states
are grown in dissimilar isotopic environments, for example, in
media containing light and heavy versions of stable amino acid
isotopes [21–23]. Equimolar quantities of light and heavy cells
are mixed, then processed, digested and analysed by MS.
The second approach uses 18O during proteolysis [24].
Digestion of a protein in 18O-enriched water results in the
incorporation of heavy isotopes into the C-terminus of each
peptide. The third method is to label a specific amino acid, for
example cysteine, with a chemical reagent containing a light or
heavy isotope. Such reagents often incorporate an isotope-
coded affinity tag, which allows enrichment of the labelled
peptides and determination of the relative abundance of the
peptides [20]. For all three methods, peptides are observed as
doublets with mass differences corresponding to the light and
heavy isotopes, and the ratio of peak height and peak area is
proportional to the relative abundance of each peptide. New
approaches for protein and peptide quantitation based on the
modification of specific amino acids with stable isotopes are
constantly emerging, as exemplified by a recent article from
Olsen et al. [11].
Issues in MS data analysis
It is now relatively straightforward to establish a high-through-
put proteomic laboratory and produce large quantities of data.
Data generation no longer represents a major bottleneck.
Nonetheless, the proteomic pendulum has swung away from
data generation and towards data validation, that is, relating the
information generated by MS and proteomics to the biological
question.
The publication of nonvalidated data sets has created confu-
sion and misunderstanding because there is an inability to
relate realistically the ‘numbers’ to a biological application. In
addition, manual validation is an extremely tedious exercise
that is highly subjective to human variability. Another major
issue in dealing with large data sets generated by LC–MS is the
existence of trustworthy software to (i) process, (ii) search, and
(iii) analyse, verify and curate the data.
S44 www.drugdiscoverytoday.com
DDT: TARGETS Vol. 3, No. 2 (Suppl.), 2004
Data processing
Before searching information generated by MS, the data must
be in a format compatible with a database search engine.
Processing software converts raw mass spectra into a generic
list of monoisotopic mass and intensity. The mass-to-charge
(m/z) ratio for each peak is defined by the centroid (i.e. the
centre of the peak mass), which is determined above a speci-
fied percentage of the peak height (e.g. 50%), and selected to
avoid the inclusion of noise at the baseline. Many of the soft-
ware programs available for transforming raw mass spectra
attempt to simplify the data by adjusting parameters such as
signal-to-noise ratio, smoothing, baseline subtraction, peak
extraction and filtering according to specific criteria that
exclude nonpeptide peaks, data centroiding, and charge-state
determination of the precursor ion. The result of such spectral
manipulation, in many instances, is that data can be omitted.
Data searching
Search engines such as Sequest [25], Mascot [26], Protein
Prospector [27] and Sonar [28] are founded on probability-
based scoring algorithms and, as such, are fraught with prob-
lems in assessing the trustworthiness of an identification. The
output from a search engine analysis is a series of peptide and
protein identifications that are ranked according to specified
criteria. Because there are no universal standards for scoring
the output from the programs, determining what constitutes a
significant match is not straightforward. If the data are of suffi-
cient quality, then the probability of a false positive can be dis-
regarded; however, in some cases the identification returned by
the search engine is likely to be incorrect.
In an attempt to increase the confidence of a protein
identification, it has become common practice to submit the
same data to two or more search engines and combine the out-
puts. The claim is that if two search engines return the same
protein, then confidence in a definitive identification will in-
crease. However, the same data are assessed with similar search
algorithms, which might exacerbate the incidence of false pos-
itives. Ideally, the data should be assessed by ‘unrelated’ algo-
rithms. It is reasonable to speculate that when different algorithms
return the same identification, confidence in the ‘correct’ protein
identity will increase.
The information returned by the search engine should thus
be taken as an initial data filter rather than the ultimate final
and correct answer. After data filtering, the remaining unin-
terpreted data should be searched against expressed sequence
tag (EST), single nucleotide polymorphism (SNP), alternatively
spliced protein databases and, ultimately, genome databases.
Exhaustive data searches would guarantee that all publicly
available sequence information is accessed and, thereby,
the incidence of false-positive identifications would be
reduced.
DDT: TARGETS Vol. 3, No. 2 (Suppl.), 2004 mass spectrometry in proteomics supplement | reviews

Data analysis and curation

Reassembly of peptide information back Protein sample Protein identifications
to the protein is a dilemma for ‘shotgun Protein
level A B C D A B C
proteomics’. Figure 1 is an overview of the
Peptide
process by which data are produced by grouping,
Enzymatic
MS and illustrates the associated difficulty digestion validation
of reassigning identified degenerate pep-
tides back to the parent protein.
Peptide
level
Potential solutions to assist the
analysis of large-scale MS data Peptide mixture Peptide identifications

sets LC/MS/MS Database

search,
From a biological point of view, improve- validation
ment in experimental design is manda-
I/MS/MS spectra
tory as more researchers are realizing the level
impact of design and methodology on
the quality of the results. For example, the MS/MS spectra

analysis of total cell lysate gives a ‘snap Drug Discovery Today: TARGETS
shot’ of the most abundant proteins,
whereas ‘hypothesis-driven proteomics’ Figure 1. Simplified outline of the experimental steps and flow of the data in a typical high-
throughput mass spectrometry (MS)-based analysis of complex protein mixtures. Each
(e.g. specific pull-down experiments or sample protein (yellow circle) is cleaved into smaller peptides (yellow squares), which can
subcellular fractionation) provides insight be either unique to that protein (unbroken arrows) or shared with other sample proteins
into the mechanism or function of a (broken arrows). The peptides are then ionized, and selected ions are fragmented to
produce tandem MS (MS/MS) spectra. Some peptides are selected for fragmentation
particular biological system. several times (broken arrows), whereas some are not selected even once. Each acquired
From an analytical angle, the quality of MS/MS spectrum is searched against a sequence database and assigned a best-matching
the data produced from the mass spec- peptide, which might be correct (yellow squares) or incorrect (black square). The database
search results are then manually or statistically validated. The list of identified peptides is
trometer is of fundamental importance.
used to infer which proteins are present in the original sample (yellow circles), and which are
Low-resolution three-dimensional ion traps false identifications (black circles) corresponding to incorrect peptide assignments. The
are extremely popular and well-suited process of inferring protein identities is complicated by the presence of degenerate peptides
to high-throughput LC–MS; however, the corresponding to more than a single entry in the protein sequence database (broken
arrows). Adapted, with permission, from Ref. [33], © (2003) American Chemical Society.
charge state of a precursor ion cannot be
distinguished when operating in full-scan
mode. The combination of a quadrupole mass selector and to process the information into a format that is compatible
collision cell with orthogonal acceleration has led to high reso- with search engine analysis. Effective automated transfer of MS
lution (~10 000) and mass accuracy (5–20 ppm with internal data to informatic programs is dependent on the reliable per-
calibration). Charge states are more readily assigned, which is formance and accuracy of peak-assignment algorithms. Most
an important advancement in minimizing false protein identi- instrument vendors provide proprietary algorithms as part of
fications. Fourier transform ion cyclotron resonance MS pro- the acquisition software, and there have been considerable ef-
vides the ultimate performance with a mass accuracy of 1–5 ppm; forts by independent groups to develop alternative mathemati-
however, such instruments are beyond the budget of most cal approaches to maximize information extraction from the
laboratories. data.
Ultimately, intelligent software solutions are vital to the contin- A lack of fully automated data analysis systems for protein
uation of proteomics. Areas of importance include (i) adapting identification from complex mixtures requires the development
current algorithms and developing new algorithms to process of more rigorous search and scoring algorithms. Recently,
and search data, (ii) maximizing information extraction, (iii) Colinge et al. [29] introduced a scoring scheme named OLAV.
semiautomatic to automatic data validation and generation of This scheme is similar to established probability-based search
statistically sound data, and (iv) data management and curation. engines, but the scoring has been markedly improved by in-
cluding structural information from consecutive fragment ion
Data processing and search algorithms matches; that is, it is an extension of the sequence tag concept.
There have been numerous attempts to determine the optimal The overall benefit of OLAV compared with other probability-
means of extracting information from MS-generated data and based search engines is the improved selection of correct peptide

www.drugdiscoverytoday.com S45
reviews | mass spectrometry in proteomics supplement
assignments based purely on the score returned by the search
engine.
Other algorithms, including Protein Prospector http://www.
ucsf.edu), ProbID [30] and Sequest [31], are under further de-
velopment with the aim of providing more reliable signifi-
cance thresholds. It still remains to be seen, however, whether
putative matches close to the threshold can be unequivocally
trusted.
Maximal extraction of information
Multiple analyses of the same sample by MS via an ‘exclusion
list’ approach [32] and iterative database analyses of the same
data are being combined in an approach to extract information
exhaustively from a single biological experiment (MDS
Denmark).
During LC–MS analysis of a mixture of proteins on a
quadrupole time-of-flight (TOF) instrument, the TOF region
can resolve significantly more precursor ions than the mass
spectrometer can select and fragment; therefore, a single LC–MS
acquisition is insufficient to analyse all precursor ions compre-
hensively. Multiple LC–MS analyses of the same sample and the
generation of an exclusion list between consecutive analyses
provide a means whereby only unique precursor ions are frag-
mented. After each LC–MS analysis, the m/z ratio and retention
time of all of the precursor ions selected for tandem MS
(MS/MS) are added to an exclusion list.These peptides are dis-
qualified from selection in subsequent analyses, and peptides
that were not previously selected are fragmented. The greater
the number of peptides selected for fragmentation by the mass
spectrometer, the greater the number of peptides that will match
a given protein, thereby increasing the sequence coverage and
the probability of a correct identification. Following data gen-
eration, the files are iteratively searched through a combination
of static and dynamic databases. The aim is to extract more
information (e.g. posttranslational modifications, alternatively
spliced proteins and point mutations) from a single experiment.
The analysis of a complex biological mixture by LC–MS usu-
ally involves the production of several fractions, each fraction
is analysed, and the protein database searched. This represents
linear data generation and analysis (Figure 2a). Alternatively,
each fraction is analysed several times by LC–MS and the data
are cycled through several databases (Figure 2b). Thus, the
quantity of data that can be generated from a single biological
experiment exponentially explodes.
Data validation and statistical analysis
An experimental peptide identification repository (EPIR) is a
recently developed database system that facilitates the valida-
tion and statistical analysis of data generated by LC–MS. One of
the software modules enables automated peptide validation. To
enhance the sensitivity of true identification and to avoid false
S46 www.drugdiscoverytoday.com
DDT: TARGETS Vol. 3, No. 2 (Suppl.), 2004
positives, additional parameters related to the identification are
computed. An important parameter is the number of sibling
peptides [33]. NSP is the total number of unique peptides
identifying a protein (or protein group) and is a strong indica-
tor of the probability that the peptide identification is correct.
Similar to OLAV, a score is calculated for consecutive y- and
b-ion fragment matches. Information concerning specific frag-
ment ions (e.g. proline), missed cleavage sites and quantitative
information (if available) are also scored. MS/MS spectra that
are rejected by autovalidation can be manually validated directly
from the raw data by generating a sequence tag, or reassessed
by the breakpoint algorithm with alternative parameters.
PeptideProphet (http://www.systemsbiology.org) [34] is
an open source software program that facilitates automatic
validation of proteomic data. Based on an empirical statistical
model, a sensitivity threshold for correct and incorrect peptide
identification can be selected. As would be expected, a higher
threshold will increase the number of false positive identifica-
tions. Thus, the gain obtained from automatic validation is lost
because of the need for additional manual validation to ensure
that the peptides and proteins returned are indeed correct.
ProteinProphet [33] is a tool for protein validation based
on the output of confirmed peptides from PeptideProphet.
Identified peptides corresponding to the same protein are
combined using a statistical model to estimate the probability
that the protein is present. The number of sibling peptides is
also estimated and the information is used to improve peptide
validation.
Another approach for peptide validation is to analyse the data
by an alternative algorithm, such as sequence tag [35], after
the initial first-round analysis using a breakpoint algorithm.
Confirmation of peptides by two unrelated algorithms will
increase the probability of protein identification.
Data management and curation
Combining all of the information generated from a large-scale
MS investigation in a simple fashion that not only allows pep-
tide and protein validation, but also grouping of the results,
reassembly of peptide information into proteins, and data re-
porting in a format that is simple to understand is not a trivial
task. There are several filtering and visualization programs that
attempt to simplify large MS data sets [36–38].
The EPIR database containing validated and confirmed pep-
tides has a series of modular interactive software tools that over-
lay the peptide database. These tools include (i) grouping of
peptides to related proteins, (ii) addition of quantitative data,
(iii) extraction of statistical information from within a single
experiment, across multiple MS analyses and different experi-
ments, and (iv) data collation into a simple format showing,
for example, protein sequence, confirmed peptides and database
accession identifiers.
DDT: TARGETS Vol. 3, No. 2 (Suppl.), 2004 mass spectrometry in proteomics supplement | reviews

(a) (b)
CELL OR TISSUE EXTRACT CELL OR TISSUE EXTRACT

SUBCELLULAR FRACTIONATION SUBCELLULAR FRACTIONATION

e.g. plasma membrane

PROTEIN/PEPTIDE FRACTIONATION PROTEIN/PEPTIDE FRACTIONATION

e.g. off-line rpHPLC

LC–MS ANALYSIS LC–MS ANALYSIS

e.g. exclusion list

DATABASE SEARCH
DATABASE SEARCH
e.g. iterative
?

DATA MANAGEMENT DATA MANAGEMENT

e.g. EPIR
?
Drug Discovery Today: TARGETS

Figure 2. Proteomic hierarchy: from cell or tissue extract to peptide or protein identification and data management. (a) Generic linear analysis by
liquid chromatography mass spectrometry (LC–MS) and database searching. Cell or tissue extracts are fractionated into subcellular components
and then further fractionated at the protein or peptide level. Each sample is analysed once by LC–MS, and the data generated are searched
against a single database. (b) Advanced exhaustive data production and database analysis. Cell or tissue extracts are fractionated into
subcellular components, such as plasma membrane, and then further fractionated at the protein or peptide level by, for example, off-line reversed-
phase high-performance liquid chromatography (rpHPLC). Each sample is analysed three times by LC–MS via the exclusion list approach, and for
each analysis the data are searched iteratively against a series of databases. The quantity of data generated from a single biological experiment
exponentially explodes through the application of multiple LC–MS analyses and multiple database searches (combined static and dynamic
databases). Peptide-centric databases for storing and mining LC/MS/MS data hold the key to large-scale MS data consolidation.

Similarly, Interact (from the Institute for Systems Biology)
[36] allows rapid and flexible interrogation and analysis of mul-
tiple data sets including filtering, unfiltering, sorting, grouping
and highlighting of the data by user-controlled criteria.
Comparisons across experiments are achieved using a tool that
highlights similarities and differences at either the peptide or
the protein level. Interact interfaces with quantitative software
to provide the average relative abundance and standard deviation
for each protein.
The potential issues outlined in this section to assist in the
analysis of large-scale MS data sets are multifaceted, highly
interdependent and cross-functional. The demand to formulate
intelligible answers from MS-generated data will continue to
direct advancement in sample preparation, MS and software
development.
Perspectives
Large-scale data production by MS and analysis of the results in
a biological context are still the domain of specialists who are
aware of the disadvantages and restrictions of the technology.
Expert knowledge is heavily exploited when considering the
limitations of an experiment and the answers obtained. As MS

www.drugdiscoverytoday.com S47
reviews | mass spectrometry in proteomics supplement
and proteomics become increasingly accessible to a broader range
of researchers – medical clinicians, for example – it is impera-
tive that there is sufficient support to assist ‘novice’ scientists in
formulating realistic conclusions from the data. Without assis-
tance, an inadequate understanding of the technology will
severely impact the reliability and significance of the results.
The future demand for software and statistical analysis of MS
data will continue to grow as increasingly more laboratories
realize the need for exhaustive data analysis rather than exces-
sive data production. Quantitation adds an extra dimension of
complexity to the data that cannot be addressed generically by
current software tools. Therefore, further software development
in this area is essential. The integration of visionary, well-
formulated biological theories with MS, plus access to simple
and comprehensible software solutions, is the key to bringing
the realm of MS and proteomics successfully within the reach
of everyday scientists.
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In the 1st May 2004 issue of Drug Discovery Today…

Editorial
Rational optimization of proteins as drugs: a new era of 'medicinal biology'.
by David Szymkowski

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