Special issue paper

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3157

Recent advances in proteomics: towards the

human proteome
Kui Wanga,b, Canhua Huanga and Edouard Niceb*
ABSTRACT: After the successful completion of the Human Genome project in 2003, the next major challenge was to
understand when and where the encoded proteins were expressed, and to generate a map of the complex, interconnected
pathways, networks and molecular systems (the human proteome) that, taken together, control the workings of all cells,
tissues, organs and organisms. Proteomics will be fundamental for such studies. This review summarizes the key discoveries
that laid down the foundations for proteomics as we now know it, and describes key recent technological advances that will
undoubtedly contribute to achieving the initial goal of the Human Proteome Organization of identifying and characterizing
at least one protein product and representative post-translational modifications, single amino acid polymorphisms and splice
variant isoforms from the 20,300 human protein-coding genes within the next 10 years. Successful unraveling of the human
proteome will undoubtedly improve our understanding of human biology at the cellular level and lay the foundations for
improved diagnostic, prognostic, therapeutic and preventive medical outcomes as we enter the era of personalized medicine.
Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: Proteomics; human proteome; HUPO; protein MS; automated sample preparation; validated antibodies

Introduction et al., 1989; Hu et al., 2005). Such mass spectrometers provide

The foundations for proteomics as we now know it were laid
down more than 30 years ago with the development by
Macfarlane and Torgerson (1976) of Cf252 plasma-desorption
mass spectrometry (PDMS) with time of flight analysis (later
commercialized as the Bio-Ion in 1982), which was used to
successfully detect insulin (Hakansson et al., 1982), followed
closely by the development of fast atom bombardment mass
spectroscopy (FABMS) techniques for protein and peptide
analysis (Barber et al., 1981; Dell and Morris, 1982). However, it
was perceived that both these technologies had limitations in
both the mass range (<25 kDa) and sensitivity (picomole) that
would ultimately be required for comprehensive analysis of
proteins and peptides in biological samples (Roepstorff, 1996),
and, in the case of the Bio-Ion, the lifetime of the Cf252 source
(half-life 2.64 years).
The next major breakthroughs came in 1988 when two
techniques, which remain the cornerstone of many proteomic
studies to this day, were published: ESI and MALDI. John Fenn from
Yale University described a new ionization technique, termed
electrospray ionization (ESI), for protein analysis (Meng et al.,
1988), and Michael Karas and Franz Hillenkamp, from the Univer-
sity of Munster in Germany, presented a new ionization technique,
matrix-assisted laser desorption/ionization (MALDI; with which
they analyzed lysozyme (Mr 14,306), β-lactoglobulin (Mr 18,277),
trypsin (Mr 23,463) and bovine serum albumen (Mr ~67,000; Fig. 1).
John Fenn was to win the Nobel Prize in Chemistry with Koichi
Tanaka in 2002 for their work on soft desorption ionization for
mass spectrometric analyses of biological macromolecules.
More recent advances in mass spectrometry include the
development of the Orbitrap and the use of ESI. Invented by
Alexander Marakov in 1999 (Makarov 2000), the Orbitrap uses
the principle of radially trapping ions about a central spindle
electrode and it can be equipped with an ESI source (Fenn
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excellent sensitivity and mass resolving power (Hardman and
Makarov, 2003). The use of peptide sequence tags (Mann and
Wilm, 1994), whereby MS/MS data is used for matching peptides
to their cognate protein, facilitated identification.
Coupled with these advances in mass spectrometry,
developments in the separation sciences were also required for
pre-purification and enrichment of low abundance proteins in
complex biological mixtures. However, while N-terminal sequence
analysis using Edman Chemistry (Edman, 1950), which had been
the method used for protein characterization prior to the develop-
ment of mass spectrometry, required purification to homogeneity,
for MS only partial purification is required.
Foremost among the protein separation techniques used for
proteomic analysis has been two-dimensional polyacrylamide
* Correspondence to: Edouard Nice; Department of Biochemistry and Molecular
Biology, Monash University, Clayton, Victoria, 3800, Australia. Email: ed.
nice@monash.edu
a
The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University,
Chengdu, 610041, People’s Republic of China
b
Department of Biochemistry and Molecular Biology, Monash University,
Clayton, Victoria, 3800, Australia
Abbreviations used: 2D-PAGE, two-dimensional polyacrylamide gel
electrophoresis; ABP, albumin-binding protein; ESI, electrospray ionization;
FABMS, fast atom bombardment mass spectroscopy; FASP, filtered aided
sample preparation; FFPE, formalin-fixed, paraffin-embedded; HUPO,
Human Proteome Organization; IMS, imaging mass spectrometry; MALDI,
matrix-assisted laser desorption/ionization; Mr, molecular weight; MRM,
multiple reaction monitoring; MWCO, molecular weight cut-off; OxICAT, ox-
idative isotope-coded affinity tags; PDMS, plasma-desorption mass spec-
trometry; prEST, protein epitope signature tag; PTM, post-translational

modification; QQQ, triple quadrupole; SRM, selected reaction monitoring.

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Recent advances in proteomics
Figure 1. The first MALDI spectrum of bovine serum albumin
(reproduced by permission of Karas and Hillenkamp, 1988).
gel electrophoresis (2D-PAGE) and microchromatography.
Patrick O’Farrell developed high-resolution methods for 2D-gel
electrophoresis in the early 1970s (O’Farrell, 1975; Fig. 2) which
are still widely used, although he apparently never originally
envisaged the impact that this technology would have on
understanding modern systems biology (O’Farrell 2008). Today,
over 10,000 spots can be separated in a single run using large
format gels (Klose and Kobalz, 1995).
In the chromatography arena, early work on the development
of micropreparative HPLC systems was initially driven largely by
the need to purify low microgram quantities of low abundance
proteins such as growth factors, their receptors and other
signaling molecules from complex biological matrices in high
yield for N-terminal sequence analysis. It was quickly realized
that the use of ‘conventional’ 4.6 mm i.d. columns gave vanish-
ingly low protein recovery levels owing largely to on-column
losses by nonspecific absorption, and that column dimensions
had to be carefully tuned to the sample load applied. By contrast
the use of short (3–10 cm) columns of reduced i.d. (1–2.1 mm)
gave excellent recovery at high concentration of microgram
quantities of protein (Nice, 1990) when operated at the
equivalent linear flow velocity. These columns could be used in
Figure 2. An early (27 May 1974) 2D-PAGE separation of Escherichia coli
14
proteins labeled with C-amino-acids (reproduced with permission of
O’Farrell, 2008).
Biomed. Chromatogr. 2014; 28: 848–857 Copyright © 2014 John Wiley & Sons, Ltd.
a multidimensional mode, giving high purification factors, for
effective sample workup prior to proteomics analysis (Nice
et al., 2007). In addition, depletion strategies (e.g. Agilent MARS;
Agilent Technologies, Inc., Santa Clara, CA, USA) and Sigma
ProteoPrep® 20 (Sigma-Aldrich Co., St Louis, MO, USA) were
developed to remove the high abundance ‘housekeeping pro-
teins’, particularly in serum, which tended to mask low-level
components during MS analysis, although it was quickly realized
that some lower abundance proteins could ‘piggy back’ on these
high abundance proteins during depletion and therefore be lost.
In addition, the peptide chromatography needed to be opti-
mized for MS compatibility, and capillary columns (<1 mm i.d.)
were developed for sample delivery to the mass spectrometer
at low flow rate (Tong et al., 1997). Currently, to achieve the
separation efficiency required for the separation of complex
peptide mixtures, high efficiency nano HPLC columns
with smaller i.d. (10–150 μm) operated at very low flow rate
(10–1000 nL/min) are being widely used (Karlsson and Novotny,
1988). Recently, Mechtler’s group developed a novel nano LC
method in which ultralong gradients (Acclaim PepMap C18; 2 cm ×
100 μm × 5 μm, 100 Å; Dionex, Sunnyvale, CA, USA) were used to
analyze the whole-cell protein extracts. In this study, 2761 proteins
from Hela cell lysate were identified (Kocher et al., 2012) in a single
run. Likewise, Mann’s group (Thakur et al., 2011) has shown
how the use of long chromatographic run times (gradient times
up to 6 h) can result in deep and highly sensitive coverage of the
yeast proteome without prior fractionation.
Clearly, advances in proteomics have, as summarized above, like
advances in genomics, been largely driven by technological
innovation. Progress in the proteomics field is exemplified by the
exponential increase in the number of proteomics publications
(Fig. 3A) and the number of human protein sequences deposited
in the databases (Fig. 3B, 3C). Until fairly recently many people
thought that proteomics had underperformed for the large
amount of capital invested, particularly in the fields of biomarker
discovery and validation and clinical proteomics (Poste, 2011;
Service, 2008; Zolg, 2006). However, the tide now appears to be
turning (Nice, 2013), and technology once again is leading the way.
The role of the Human Proteome
Organization
It is now realized (Hood et al., 2012; Poste, 2012) that big-science
projects like the Human Proteome Project require careful coordi-
nation to bring together the requisite global multidisciplinary
teams and infrastructure (e.g. protein chemists, peptide
chemists, organic chemists, molecular biologists, physicists,
clinicians, bioinformaticians, statisticians, computer scientists,
engineers, regulatory experts, instrument companies, diagnostic
companies and well curated biobanks) required for success. The
Human Proteome Organization (HUPO) was founded in 2001
with the goals of promoting the field of proteomics and its
application through international cooperation and research
collaborations and, importantly, by fostering the development
of new technologies and techniques and the necessary reagents
(Omenn, 2013).
HUPO has identified three working pillars – mass spec-
trometry, antibodies and an integrated knowledge base
(bioinformatics) – and has structured discovery around
chromosome-centric (C-HPP) and disease and biology-based
approaches (B/DHPP; Fig. 4; Legrain et al., 2011). The primary
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Figure 3. Proteomics publications and human protein sequences deposited in the databases. The number of proteomics publications per annum. The
data was generated by performing a Pubmed search with the term ‘proteome’ or ‘proteomics’ in the title or abstract (A). The number of human protein
sequences that has been deposited to Swiss-Prot (B) or TrEMBL (C). The primary data was downloaded from http://www.uniprot.org/
Figure 4. The Human Proteome Organization (HUPO) operating structure
(reproduced by permission of Legrain et al., 2011). Note the three pillars:
mass spectrometry (MS), antibodies (Abs) and knowledge base (KB).
goal of HUPO is to identify and characterize at least one protein
product for each of the 20,300 human protein-coding genes
including examples of post-translational modifications (PTMs),
single amino acid polymorphisms and splice variant isoforms. At
present about 5000 (25%) of the predicted human protein-coding
genes lack any experimental evidence at the protein level (Gaudet
et al., 2013). Providing confirmatory evidence of these putative
proteins is an initial HUPO priority. Some of the key recent technol-
ogy advances that will assist in the unraveling of the human
proteome are summarized below, with examples from the litera-
ture and our own laboratories.
Multiple reaction monitoring
The development of targeted sensitive and specific label-free
quantitative techniques compatible with protein and peptide
analysis such as multiple reaction monitoring (MRM, also known
as selected reaction monitoring, SRM), capable of the multiplex
analysis of large numbers of targets in a single MS run, has
significantly advanced our understanding of the human
proteome, especially for discovery of biomarkers of human
disease. Various types of instruments capable of two stages of
mass filtering can be used for MRM assays, although typically a
triple quadrupole (QQQ) instrument is used. Specific proteotypic
peptides generated by enzymatic degradation of a protein
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K. Wang et al.
sample (typically using trypsin) are introduced by HPLC into
the first quadrupole of the QQQ instrument where proteotypic
precursor ions are preselected and, following collision-induced
dissociation of the precursor ions in the second quadrupole,
selected product ions are monitored in the third sector. MRM
has the advantage over antibody-based techniques that it is
generic and thus obviates the need to generate expensive
reagents for each individual assay. The complexity of the resulting
MRM spectra is significantly reduced (Fig. 5), facilitating the detec-
tion and measurement of low abundance proteins. If scheduled
MRM is used, in which selected ions are only monitored during
the time window in which they elute from the HPLC (Lange
et al., 2008), multiplex analysis of hundreds of targets is possible.
Selectivity is afforded from the mass of the specific proteotypic
peptide chosen, the corresponding product ions and the charac-
teristic HPLC elution time. The sensitivity is now approaching that
of ELISA assays, particularly if specific enrichment techniques are
used (see later).
Quantitative MRM methods have been developed for analysis
of samples from a number of biological matrices, including
blood (Anderson and Hunter, 2006; Domanski et al., 2012;
Stahl-Zeng et al., 2007), urine (Chen et al., 2012) and feces (Ang
and Nice, 2010; Ang et al., 2011). In the case of the fecal samples
it was possible to analyze carcinoembryonic antigen, a low level
component, in trifluoroacetic acid fecal extracts (ng/mg stool),
without prior sample fractionation (Ang et al., 2011).
SWATH
Another important technological advance that will facilitate
consistent and accurate proteome analysis is SWATH (Sequential
Windowed data independent Acquisition of the Total High-reso-
lution mass spectra; Gillet et al., 2012). In SWATH mode, instead
of the Q1 quadrupole transmitting a narrow selected mass range
through to the collision cell, a wider window containing multiple
analytes is allowed through. This generates a complex MS/MS
product ion spectrum containing data on all the analytes
contained within the Q1 m/z window (Fig. 6). High-quality
extracted ion chromatograms (XICs) can be then generated
post-acquisition to produce MRM-like data. The Q1 window
can be stepped across the full mass range (25 m/z unit
increments), with collection of full-scan composite MS/MS
spectra at each step, within an LC compatible cycle time,
enabling a data-independent LC workflow. Importantly, the
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Recent advances in proteomics

Top-down proteomics
Another area that is rapidly advancing is top-down proteomics for
the analysis of intact proteins. Improvement of both the speed
and the quality in the individual steps required for top down
proteomics analysis now allows robust implementation for the
analysis of proteins below 70 kDa. Thus, improved intact protein
separations coupled to state-of-the-art high-performance instru-
mentation (e.g. Fourier transform ion cyclotron resonance or
Orbitrap instruments) are enhancing both the quality and the
number of proteins and protein isoforms that can be detected.
In a recent study 1220 proteins (yielding over 5000 proteoforms)
were identified from a human lung carcinoma cell line, including
300 membrane proteins (Catherman et al., 2013). Importantly,
unlike bottom-up protocols where, owing to enzymatic digestion,
detailed information on PTMs and sequence variants is
compromised, in top-down approaches intact proteins are
analyzed, allowing unequivocal identification and location of
specific modifications.

Figure 5. A comparison of Total Ion Chromatogram (TIC) and multiple
reaction monitoring (MRM) analysis of the same RP-HPLC purified fecal
sample from a patient with colorectal cancer. The proteins targeted for
the MRM analysis, in order of elution, were hemoglobin, haptoglobin and
carcinoembryonic antigen.
data can be re-mined ad libitum for alternative targets, or using
different product ions or peptides if there is signal overlap.
SWATH has recently been used to quantitate N-linked glycopro-
teins in human plasma with low ng/ml sensitivity (Liu et al., 2013),
using a hydrazine-based sample preparation protocol for glyco-
protein enrichment.
Antibody-based techniques
Good quality, validated antibody reagents, both polyclonal and
monoclonal, are an essential part of the modern proteomics
toolbox, although unfortunately many researchers have been
badly burnt by reagents of inferior quality supplied by some
manufacturers (Colwill and Graslund, 2011). It must be remem-
bered, however, that antibodies are a case of ‘horses for courses’,
and an antibody that is excellent for immunoprecipitation, for
example, may not work for immunohistochemistry. Luckily,
there are now efforts to ensure that validated antibodies against
a defined target can be chosen for a specific application.
Antibodypedia is a free, open-access, resource for data and
commentary on antibodies from both commercial and academic
providers (http://www.antibodypedia.com/). The November
2012 release catalogs more than 500,000 individual antibodies,
covering 88% of the proteins encoded by the human genome.
Antibodypedia also details nearly 145,000 experiments
performed with these antibodies, as well as 32,000 links to
publications related to their use. Side by side comparison of
the key properties of antibodies to specific targets can be made.

Figure 6. Schematic of SWATH Workflow (courtesy of Dr Christie Hunter, AbSciex). Unlike MRM, in which a narrow window is set for Q1, in SWATH a
wider window containing multiple analytes is passed. This produces a MS/MS spectrum that is a composite of all the analytes within the Q1 m/z
window. High-quality extracted ion chromatograms (XICs) can be generated post-acquisition to produce the MRM-like data. The Q1 window can be
stepped across the mass range, collecting full-scan composite MS/MS spectra at each step, enabling data-independent LC workflow.
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In collaboration with the HUPO effort, The Human Protein Atlas,
launched in 2005, was set up to allow for a systematic explora-
tion of the human proteome using an antibody-based approach
(Uhlen et al., 2010). The goal was to generate at least one
antibody to all approximately 20,300 genes in the human
genome by the end of 2014. Version 11.0, which was released
on 11-03-13, now covers 15,156 genes with protein expression
profiles based on 18707 antibodies. Key to this approach is the
use of protein epitope signature tags (prESTs) (Agaton et al.,
2003; Larsson et al., 2006) as immunogens. prESTs contain
selected proteotypic fragments, typically 25–150 residues, of
the target protein. They are produced in Escherichia coli as fusion
proteins with an N-terminal His6-ABP tag: the His6 tag allows
single-step protein purification under denaturing conditions
while the albumin-binding protein (ABP) both helps promote
an immune response and facilitate quantitation following
purification. Polyclonal antibodies are raised in rabbits and
immunopurified against the corresponding prEST. It is now
generally recognized that the rate-limiting step in the high-
throughput antibody purification is the generation of the target
antigen (either as a native protein, recombinant protein or
synthetic peptide): prESTs obviate this and offer a generic
method to rapidly obtain immunogens against all the proteins
encoded by the human genome. While polyclonal antibodies
are cheaper to produce and recognize multiple epitopes that
can be advantageous for some applications (e.g. secondary anti-
bodies in sandwich ELISAs), they suffer the disadvantages of
finite supply with each batch requiring fresh validation. Mono-
clonal antibodies, by contrast, are a renewable resource where
all batches are identical, generally have lower nonspecific
binding than polyclonals, and have a single epitope. This has
stimulated the development of high-throughput methods
capable of meeting the global monoclonal antibody demand
(Colwill and Graslund, 2011), including the use of a robotic
approach (Layton et al., 2013).
Antibody-based techniques are complementing MS approaches
(immuno-MS) in two main areas: ensuring highly specific targeting
and enrichment for enhanced selectivity and sensitivity; and
supporting targeted interactomics studies (see below). A key
example of the first approach is stable isotope standards and
capture by anti-peptide antibodies (SISCAPA; Anderson et al.,
2004). In this approach specific anti-peptide polyclonal antibodies,
generated in rabbits, are immobilized onto 100 nL affinity columns
to enrich the corresponding target peptide present in a tryptic di-
gest, along with the corresponding spiked stable-isotope-labeled
internal standard. Following elution from the anti-peptide
antibody supports, mass spectrometry is used to quantitate the
peptides (both endogenous and labeled). The method was shown
to be compatible with direct multiplex analysis of peptides
representing 47 high to medium abundance proteins in human
serum using MRM (Anderson and Hunter, 2006). The method has
now been modified to use rabbit monoclonal antibodies and
automated solid phase extraction MS/MS with a sample cycle time
of approximately 7 s (Razavi et al., 2012).
Interactomics
In living systems most proteins function by interaction with
other proteins in either stable or dynamic complexes. The sum
of these interactions in a specific biological system is the
interactome. While the yeast two-hybrid system was the original
mainstay of this technology (Lievens et al., 2010), advances in
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K. Wang et al.
proteomics, particularly the use of immunoprecipitation, pull-
downs, epitope tagging (which circumvents the need for spe-
cific antibodies against each target), tandem affinity purification
(Puig et al., 2001) and chemical cross linking (Rigaut et al., 1999),
are now becoming predominant (Braun 2013). Several early
proteomics studies focussed on yeast as a model organism
(Gavin et al., 2002, 2006; Ho et al., 2002; Krogan et al., 2006).
Collectively, these studies covered almost 50% of the yeast
proteome (Lievens et al., 2010). Importantly, the global land-
scape of the membrane interactome in Saccharomyces cerevisiae
has been characterized (Babu et al., 2012). More recently multi-
ple studies have focussed on the human interactome
(Havugimana et al., 2012). However, it was frequently noted that
the overlap between datasets was poor (Braun, 2013; Lievens
et al., 2010), possibly owing to the heterogeneity of both the
experimental systems and the processing pipelines.
Fortunately, it has now been shown (Varjosalo et al., 2013)
that the use of carefully designed standard operating proce-
dures and bioinformatics approaches can overcome this lack
of reproducibility, even between different laboratories. In this
study, the interactomes of 32 human kinases (which were
used as baits), containing 377 unique proteins and 488
interactions (Fig. 7), were determined.
Lambert et al. (2013) have recently used affinity purification
coupled with SWATH to characterize changes in protein–protein
interactions imparted by the hsP90 inhibitor nVP-AuY922 or mel-
anoma-associated mutations in the human kinase cdK4 using
pull down with Flag M2 magnetic beads followed by on-bead
tryptic digestion.
Redox proteomics
Thiol groups on cysteine residues of certain proteins (redox sensors)
can be direct targets of reactive oxygen species and can be oxidized
to sulfenic acids and further to sulfinic acids and sulfonic acids
(Leonard and Carroll, 2011). These redox modifications can act as
an on/off switch for protein activity controlling many important
biological events such as gene transcription, translation and protein
folding, cell metabolism, signal transduction, cell cycle and cell
death (Brandes et al., 2009; Paulsen and Carroll, 2010; Wouters
et al., 2011). Characterization of the redox state of redox sensors is
urgently required in the understanding of cancer biology (Parri
and Chiarugi, 2013) and stem cell biology (Wang et al., 2013).
Oxidative isotope-coded affinity tags (OxICAT), a thiol-trapping
proteomic technique, was developed by Ursula Jakob’s group to
characterize the redoxome in vivo. In this study, the reduced
thiol groups were labeled with light 12C-ICAT reagents under pro-
tein-denaturing conditions and oxidized thiol groups were labeled
with heavier 13C-ICAT. The proteins were then subjected to trypsin
digestion, affinity chromatography enrichment and LC/MS/MS
identification (Leichert et al., 2008). Using this method, they success-
fully produced a snapshot of the in vivo thiol modification status of
cellular proteins in E. coli (Leichert et al., 2008) and Caenorhabditis
elegans (Knoefler et al., 2012). It is believed that OxICAT will be
widely used to visualize the thiol status of proteins in many different
human disease states. In addition, several other proteomic-based
technologies, such as nonreducing/reducing ‘diagonal’ polyacryl-
amide gel electrophoresis (Cumming et al., 2004), quantitative gel-
based redox proteomics (Izquierdo-Alvarez et al., 2012; Leichert
and Jakob, 2004), OxMRM (Held et al., 2010), purification of
reversibly oxidized proteins (Templeton et al., 2010; Victor
et al., 2012) and the use of sulfenic acid-specific antibodies
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Recent advances in proteomics

Figure 7. The interactomes of 32 human kinases determined by affinity purification mass spectrometry (AP-MS) (reproduced by permission of
Varjosalo et al., 2013). The bait proteins used for affinity purification are shown in the rectangles. The thick lines correspond to interactions present
in the public databases. Black dashed lines indicate known interactions between the proteins identified by AP-MS.

(Seo and Carroll, 2009) and S-glutathionylation-specific antibodies
(Yusuf et al., 2010), have extended the boundaries for detecting
and quantifying redox cysteine modifications in a cellular context.
Imaging mass spectrometry
Imaging mass spectrometry (IMS) allows region-specific mea-
surement of proteins and peptides directly from biological
specimens (e.g. tissue slices) immobilized on a MALDI target,
allowing detection of analytes in situ without the need for
labeling (Cornett et al., 2007). The ability to carry out multiplex
analysis of hundreds of analytes in a single experiment without
prior knowledge of tissue composition is most attractive. Funda-
mental to this technology is sample preparation. Unique to
imaging mass spectrometry compared with other mass spectro-
metric techniques is that preparation of the sample and
acquisition of the MS data must be performed in such a way
as to preserve the spatial integrity of the sample within the limits
of the spatial resolution of the measurement (Norris and Caprioli,
2013). Archival formalin-fixed, paraffin-embedded (FFPE) tissues
offer an invaluable source of potential retrospective proteomic
information for such studies (Lemaire et al., 2007), with the
associated clinical data readily available. However, analysis of
archival FFPE tissues by high-throughput proteomic methods
has been hindered by the adverse effects of formaldehyde
fixation and subsequent histological processing resulting in
chemical modification of the target proteins, in particular
methylol (hydroxymethyl) adducts and stable cross-linked
Schiff’s bases and methylene bridges (Fowler et al., 2013), and
significant effort has been made in recent years to overcome
these problems. It has been found that the use of antigen-
retrieval techniques, similar to those used in immunohistochem-
istry (Gustafsson et al., 2010), is a possible solution.
A number of interesting clinical applications of IMS have been
reported. These include studies on the molecular physiology of
the eye, determination of human epidermal growth factor
receptor 2 (HER2) status in cancer tissues, neuroscience and
drug analysis (Norris and Caprioli, 2013).
Automated sample preparation
The need, and the ability, to rapidly analyze large numbers of
biological samples by mass spectrometry in a high-throughput
format have stimulated the need to develop compatible
automated methods of rapid sample preparation. Automated
MALDI plate spotting was one of the earlier applications where
eluent from HPLC columns was collected directly onto the
MALDI plates. Automated techniques have been also been
developed using chromatographic column, microplate and
magnetic bead-based formats, and multidimensional protocols
can readily be incorporated. Typically some form of robotic
liquid handling platform is utilized. Thus, Kreusch et al. (2008)
combined size exclusion, anion exchange and lectin affinity
chromatography orthogonally using a robotic system developed
‘in house’ to analyze human serum. This system was used to
search for biomarker candidates in sera of patients with Alport
syndrome and severe inflammation. Twenty-three new marker
candidates were detected for Alport syndrome and 33 new for
severe inflammation (Baum et al., 2008). In another approach,
Loo et al. (2010) developed a panel of seven Lectin-Dynabeads
for the effective one-step purification of serum glycoproteins in
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96-well microplate format. This method is also being used for
biomarker discovery.
Filtered aided sample preparation (FASP) (Wisniewski et al.,
2009), is another technique applicable to high-throughput
sample preparation, using the principle of protein retention
and buffer exchange by ultracentrifugation on molecular weight
cut-off (MWCO) spin columns to enable protein denaturation,
reduction, alkylation and digestion without sample transfer.
FASP has been multiplexed in 96-well filter plate format (Switzar
et al., 2013) making it amenable to robotic automation. Cells or
tissues can be lysed in a 96-well plate and then transferred
directly to a 96-well MWCO filter plate for simultaneous process-
ing. In addition to cells and fresh tissues, a recent study showed
that proteins extracted from FFPE tissues can also be applied to
FASP (called FFPE-FASP; Ostasiewicz et al., 2010).
As another alternative, combinatorial hexapeptide ligand
libraries (CPLL), containing millions of different ligands, have
been used in an affinity saturation strategy to amplify low abun-
dance proteins using what has been termed ‘equalizer
technology’ (Di Girolamo et al., 2011). The library is bound onto
solid-phase porous beads, and is now commercially available
(ProteoMiner, Bio-Rad, Bio-Rad Laboratories Inc., Hercules, CA,
USA). This approach has recently been coupled with MRM (Di
Girolamo et al., 2011) using Saccharomyces cerevisiae as a model
system. A set of target proteins covering a broad abundance
range was selected to quantitatively evaluate the precision of
the approach and its capability to detect low-abundance proteins.
A number of instrument companies are now developing
platforms for high-throughput sample preparation. Agilent
Technologies have developed their AssayMAP Bravo Platform
and affinity cartridges that allow 96 samples to be processed
simultaneously. PhyNexus (PhyNexus, Inc., San Jose, CA, USA)
have developed a range of ‘in tip’ columns (PhyTip) compatible
with their own micro-extractor automated instrument (Le et al.,
2013) as well as other robotic platforms such as Tecan and
Hamilton. Atoll GmbH (Weingarten, Germany) also produce a
range of RoboColumns (www.atoll-bio.com) in 96-array format
which can be packed with any chromatographic support.
Shimadzu (Tokyo, Japan), taking an automated chromatography
approach, market the Perfinity system that integrates automated
affinity selection, buffer exchange, digestion, desalting and
reversed-phase separation, allowing the generation and
purification of peptides from serum for mass-spectral analysis
in approximately 10 min. It should be noted that robotics may
also be used to rapidly optimize chromatographic and affinity
elution conditions.
Bioinformatic tools for data analysis
Data processing is undoubtedly a critical component in any
proteomics experiment, and is frequently the most time-
consuming. Recent years have seen significant advances in the
field of proteomics-related bioinformatics and, in addition to
the proprietary software supplied by many of the instrument
manufacturers, many free and sophisticated open-source soft-
ware tools are currently available (Deutsch et al., 2008a).
Examples include Proteome Discoverer (Thermo Fisher Scientific
Inc., Waltham, MA, USA), SIEVE Software (Thermo Fisher Scientific
Inc.), Mascot Daemon (Matrix Science, London, UK), MaxQuant
(http://www.maxquant.org/), the Trans-Proteomic Pipeline, a suite
of software tools for the analysis of MS/MS datasets (Deutsch et al.,
2010), Skyline (https://skyline.gs.washington.edu/labkey/project/
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K. Wang et al.
home/software/Skyline/begin.view) for SRM/MRM and SWATH
quantitative analysis, freely accessible protein databases of protein
sequence and functional information such as UniProt (http://www.
uniprot.org/) and programs such as KEGG (http://www.genome.jp/
kegg/pathway.html), STRING (http://string-db.org/), Cytoscape
(http://www.cytoscape.org/) and Ingenuity (http://www.ingenu-
ity.com/) for pathway analysis, and gene ontology analysis tools
such as the Gene Ontology (http://www.geneontology.org/),
GOEAST (http://omicslab.genetics.ac.cn/GOEAST/) and DAVID
Bioinformatics Resources (http://david.abcc.ncifcrf.gov/).
Databases/browsers
High-throughput proteomic experiments generate huge amounts
of peptide data, much of which has been stored and dissemi-
nated in multiple databases, such as PeptideAtlas (Deutsch
et al., 2008b), PRIDE (Vizcaino et al., 2010), Human Proteinpedia
(Kandasamy et al., 2009), GPMDB (Craig et al., 2004), neXtProt
(Lane et al., 2012) and Human Protein Atlas (Berglund et al.,
2008). To ensure rapid and open sharing of proteomic data,
policies for data deposition are being implemented by the
leading journals in the field (Kinsinger et al., 2011). The proteomics
community is taking steps to ensure that data are made publicly
accessible and are of high quality, a challenging task that requires
the development and deployment of methods for measuring and
documenting data quality metrics. The plethora of data that
resides in these databases needs to be collated into coherent
and searchable browsers, that are regularly updated, for their
effective interrogation [e.g. to identify ‘missing proteins’, defined
as those proteins which have only transcriptomic evidence and
a predicted sequence (or are inferred by homology), or those
partially identified proteins, in which transcript evidence for the
existence of the corresponding protein is available but lacks
convincing MS information (Paik et al., 2012)]. To this end, several
browsers are currently being developed as part of the HUPO
initiative, including The Proteome Browser (Goode et al., 2013)
and CAPER (Guo et al., 2013).
Conclusions
We are now entering an era where recent advances in
proteomics, coupled with genomics, transcriptomics and bioin-
formatics, will rapidly populate the human genome. This will
lead to improved understanding of human biology, both in the
normal and the diseased state, which will bring with it enormous
economic benefits (Hood et al., 2012). This heralds the arrival of
personalized medicine, which will have an increased focus on
wellness rather than disease and which will revolutionize
treatment decisions guided by the patient’s unique circum-
stances and characteristics based on the individual’s specific
genome and proteome.
However, as pointed out in a recent article by George Poste
(2012), such a path is not without obstacles. In particular,
multidisciplinary teams involving multi-institution cooperation
will be required. The roles of bodies such as HUPO will be
imperative for co-ordinating this. In addition, significant funding
will be required to support this big science big data initiative,
involving a major paradigm shift in how funds are allocated,
without which the cohesion required for such a ‘big science
big data’ project will not be possible.
Biomed. Chromatogr. 2014; 28: 848–857
Recent advances in proteomics
Acknowledgments
We gratefully acknowledge Dr Alun Jones (University of Queens-
land) who performed the assay shown in Fig. 4. Edouard Nice was
supported, in part, by NHMRC project grant 603130, 1017078 and
development grant 1017078. In this special issue I would particularly
like to acknowledge both over 35 years of scientific interaction with
Chang Kee, and, most importantly, our long-term friendship.
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