BBA - Proteins and Proteomics 1867 (2019) 9–16
Contents lists available at ScienceDirect
BBA - Proteins and Proteomics
journal homepage: www.elsevier.com/locate/bbapap
Gel-based proteomics in disease research: Is it still valuable?☆
Yong-In Kim
a,b
, Je-Yoel Cho
a,⁎
T
a
Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine,
Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
b
Center for Bioanalysis, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Yuseong-gu, Daejeon 43113, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords:
Gel-based proteomics
Mass spectrometry
GeLC-MS
2-DE
DIGE
Gel electrophoresis had been the primary method in proteomics. In the early era of proteomics, gel electrophoresis was a dominant technique of sample preparation for mass spectrometry analysis. Particularly, twodimensional electrophoresis provided high-resolution proteome separation, and was regarded as the standard
methodology for the separation of wide-range proteomes. However, gel electrophoresis turned downwards due
to the progress of other separations including liquid chromatography and ionization techniques, resulting gelfree proteomics finally becoming dominant players at present. There are numerous advantages in gel-free approach in aspects of current trends of disease research. Interestingly, gel-free approaches are still advanced, it
seems that gel electrophoresis will not be disappeared. The unique features of gel electrophoresis can be complementary for gel-free and it is suitable for the new wave of top-down functional proteomics.
1. Introduction
mixtures due to its low mass ranges, non-proteinaceous materials, and
particularly common buffer components (also known as salt) [4].
Moreover fueled by its increasing sensitivity and ease of use, MALDI-MS
took an early lead in proteomics, yet required pre-fractionation for biospecimen measure to reduce complexity [4,6–8].
At the beginning of the time when MALDI-MS was popular, twodimensional gel electrophoresis (2-DE) was the most high-resolution
proteome separation method and was regarded as the standard methodology for the separations of complex proteomes [9]. While MALDI
did not provide any separation of components in preparative sense, it
did well measure proteins extracted from polyacrylamide gel. The
combination of 2-DE and MALDI-MS opened a new prospect in the field
of proteome research [10]. The emerging field of proteomics has grown
out of the mature technology of high-resolution 2-DE for protein separation and quantification [11]. It is commonly assumed that 2-DE-MS
is a suitable technology for global proteome analysis, based on its
ability to display, quantify and identify thousands of proteins in a single
gel [12]. However, initial dominant MALDI techniques could not last
forever, and as a result ESI finally became dominant.
ESI was being optimized to nano flow rate (nanoliter ranges per
minute) sample induction to achieve enough sensitivity for peptide
Biomarkers have been in high demand for disease diagnosis and
therapeutics. Traditional hypothesis-based research was intolerable for
massive screening works. Together with omics technologies emerged,
nowadays, the paradigm for disease research has been moving toward
evidence-based large-scale discovery studies. Proteins as key effector
molecule have been regarded ideal biomarkers for various diseases for
they catalyze every biological function [1]. Proteomics, which is represented by mass spectrometry (MS) technologies, stands as a solver
for disease diagnosis and drug target discovery.
By the 1980s, proteins and other macromolecules were hardly being
analyzed by MS. There had been no efficient soft ionization method to
transform large molecules into the gas phase without extensive fragmentation and decomposition, until matrix-assisted laser desorption
ionization (MALDI) and electrospray ionization (ESI) methods were
developed. These ionization techniques revolutionized biological MS
and are still the dominant forms of macromolecule ionization up to this
day [2]. At the beginning period of protein MS analysis era, both
MALDI and ESI techniques were initially utilized to protein ionization
[2–5]. Rather than MALDI, ESI was not used prevalently for complex
Abbreviations: MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; ESI, electrospray ionization; 2-DE, two-dimensional gel electrophoresis;
HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DIGE, difference gel electrophoresis; GeLCMS, gel electrophoresis LC-MS; BAC, bis-acrylcystamine; PTM, post-translational modification; ABPP, activity-based protein profiling
☆
This article is part of a Special Issue entitled: Electrophoresis in proteomics.
⁎
Corresponding author at: Department of Biochemistry, College of Veterinary Medicine, Seoul National University, Room 811, 1 Gwanak-ro, Gwanak-gu, Seoul
08826, Republic of Korea.
E-mail address: jeycho@snu.ac.kr (J.-Y. Cho).
https://doi.org/10.1016/j.bbapap.2018.08.001
Received 31 March 2018; Received in revised form 30 June 2018; Accepted 9 August 2018
Available online 15 August 2018
1570-9639/ © 2018 Elsevier B.V. All rights reserved.
BBA - Proteins and Proteomics 1867 (2019) 9–16
Y.-I. Kim, J.-Y. Cho
staining techniques. The choice of staining methods may offer different
sensitivity [26,27] and enable nicely visualized phosphorylation (Pro-Q
Diamond) or glycosylation (Pro-Q Emerald) (Table 1) [28,29]. Protein
stains not only give qualitative information, but also quantitative information by its density. Difference gel electrophoresis (DIGE) is protein-labeling methods using reactive fluoresce dyes (Cy2, Cy3, Cy5) to
distinct two or three samples. DIGE is free from gel-to-gel variation, and
allows the quantification and visualization of about 1500–2500 protein
spots from a gel [25,26,30,31].
Mostly selected excised protein spots are subjected to in-gel digestion for MS analysis. In-gel digestion has several following advantages.
i) simple and cost-effective procedure for pre-fractionation; ii) contaminants and detergent removal; iii) qualitative assessment via visualization; iv) capable of various type of samples [32]. It is undeniable
that 2-DE is one of the most powerful protein separating and qualitative
methods.
On the other hands, 2-DE has several weaknesses. 2-DE has a nice
capability to separate proteomes, however, some proteins are not applicable. Specific classes of protein have long been known to be excluded or under-represented in 2-DE patterns. These include highly
acidic or basic proteins, extremely high/low molecular weight proteins,
and membrane proteins [31]. 2-DE has a limit to loading capacity about
100 μg of proteins onto a single gel, and if using DIGE to compare three
samples, the amount of protein loaded per sample is reduced (~33 μg).
It should be noted that load limitation makes it difficult to detect lowabundant proteins [27,33]. The dynamic range of gel staining is restricted by densitometric platform. To overcome quantification limits,
the advent of better imaging platforms is desired [26,27,34]. Even
though proteomes were separated to two distinct characteristics, comigrated spots prevent unambiguous identification of proteins including post-translationally modified proteins and impairs the accuracy
of comparative quantification [35,36]. Without robotic spot picking,
post-staining with visible dye under the naked eye will be needed for
manual spot excision that would be possible to make slight difference of
stainability or location changes between pre- and post- stain spot
[26,27,37,38]. Additional weaknesses of gel separation are gel-to-gel
variation, expensive dyes, uncertain recovery from a gel piece, and lowthroughput identification due to hardly automated procedure
[27,31,34–36,39].
Fig. 1. Publication counts related to “gel-based” and “gel-free” proteomics. The
number of publication was quoted from PubMed (https://www.ncbi.nlm.nih.
gov/pubmed/). For “Gel-based” publication count search, following keyword
combinations used. (“gel electrophoresis” or PAGE or 1D or “1-D” or “2-D” or
“2-DE” or “2-D”) and (proteomics or proteome or proteomic). For “Gel-free”
publication count search, keyword combinations used are below. (“LC-MS” or
“liquid chromatography”) and (proteomics or proteome or proteomic).
measure in biosamples [13]. Thus, ESI overcame the limit of sensitivity
[7]. High-performance liquid chromatography (HPLC) was coupled
with the ESI-MS for the molecular fractionation prior to MS analysis
[14]. Thus, HPLC-ESI-MS had become a very powerful technique capable of analyzing various molecules in a complex biological sample
mixture with convenience and speed [3,15]. ESI became the most
popular ionization technique so far for proteome analysis [16–18].
While LC-ESI-MS went like hot cakes, MALDI-MS was at an ebb. Together with these trends, the proteomics paradigm changed from protein-centric to peptide-centric analysis (also known as bottom-up proteomics). (Fig. 1).
What were the pros and cons of each technology during the transition of Gel-based proteomics to gel-free proteomics?
2. Gel-based proteome analysis
2.1. Traditional 2-DE based proteomics
2.2. GeLC-MS: increased throughput
O'Farrell and Klose introduced modern 2-DE in 1975 that combined
the separation according to the charges by isoelectric focusing under
denaturing conditions with the fractionation corresponding to the sizes
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) [11,19]. This separation technique provided a suitable resolution of protein mixture and became the standard methods for proteome
analysis [9]. Highly separated proteins through 2-DE should be visualized by gel staining [20] followed by gel spot, which include proteinof-interest, excised and subjected to in-gel digestion by an endopeptidase (e.g., trypsin, lys-C, glu-C, chymotrypsin, etc.). The resulting peptides generated by trypsin were highly capable of ionization
and measurable within MS dynamic range [21–23]. The peptides were
measured by MS via MALDI or ESI. Tandem mass spectra derived from
fragmented peptide ion in the mass spectrometry were unique identifiers for amino acid sequence information (Fig. 2).
Because analytes are selected from a gel, 2-DE based proteomics are
extremely dependent on 2-DE quality rather than MS. High-resolution
gel separation at the protein level makes it possible to differentiate
isoforms or post translational modifications, such as glycosylation,
phosphorylation, and methylation [24–26]. With this separating power,
the fascinating feature is that the results of selectivity is several thousand protein species with low complexity. Certain spots allow direct
protein measure by MS, also called top-down proteomics, from complex
biosamples. Moreover, 2-DE gels can store the separated intact proteins
for several months [24]. Sensitivity of 2-DE-MS is dependent on
The improvement on peptide ionization efficiency of ESI spurred
development of simple and robust sample preparation methods [7]. By
nature, it is easy to combine ESI with LC, which has a good advantage of
automated separation. Typical gel electrophoresis-LC-MS (GeLC-MS) is
composed with one-dimensional SDS-PAGE and nano-flow LC-MS.
GeLC-MS combined advantages of in-gel digestion method with online
LC separation [32]. It is technically simple and combines decent protein
separation capability that also captures those proteins typically not
accessible via 2-DE (Fig. 2) [40]. The efficient and effective procedure
demonstrate that it is possible to identify proteins more than a thousand
in early 2000s [41,42], and is still used to generate large datasets of
global proteomes [43].
High throughput fractionation technique establishing offered to
develop various quantitative methods, which can provide better sensitiveness, broaden dynamic range, and high accuracy. Chemical labeling
using stable isotope (e.g. dimethyl) or isobaric tags (e.g. iCAT) which
are simple and quick methods compatible from in vitro to clinical disease samples [44–47]. Metabolic labeling (e.g. SILAC and AHA) can
reach higher labeling efficiency than others [48,49]. 2-DE based proteome quantification is analyzed on the gel via densitometric image.
However, quantification techniques described above can be utilized
only when measured by MS and/or tandem MS. So, mass difference
based quantification technique in GeLC-MS is close to gel-free fractionation rather than traditional gel separation of protein.
10
BBA - Proteins and Proteomics 1867 (2019) 9–16
Y.-I. Kim, J.-Y. Cho
Fig. 2. General workflow of gel-based proteomics. (a) Proteins are extracted from bio specimen. (b) Extracted protein mixture from biosamples separated by 2-DE or
SDS-PAGE. In most case, proteins are quantified on a gel. Using the quantitative difference from DIGE, target spots can be selected from 2-DE. (c) Excised gel pieces
are trypsinized and resulting peptides are collected. (d) Peptides are ionized via MALDI or nano ESI and are inducted to MS. (e) Peptide is measured in MS spectrum,
followed by selected and isolated, subsequently fragmented to get the sequence information from MS/MS spectrum.
3. Gel-free proteomics: convenient and in-depth proteome
coverage
the demand for in-depth proteome coverage with large-scale samples
has arisen [50]. However, particularly on a 2-DE, gel-based proteomics
need technically labor-complex and therefore cost-intensive and fundamentally limited [6]. Alternative simple methods for large-scale
study have been developed and demonstrated that LC-ESI-MS systems
can handle highly complex peptide mixtures without gel-separation
As shown in Fig. 1, the dominant player of proteomics today is gelfree. Then, what makes 2-DE to fall behind? Becoming MS powerful
technology for the analysis of large numbers of endogenous proteins,
11
BBA - Proteins and Proteomics 1867 (2019) 9–16
Y.-I. Kim, J.-Y. Cho
its modified methods allow high-throughput analysis of detergent dependent analytes like a membrane protein [59,60].
There are several modification of PAGE by changing buffers. Native
PAGE, which use non-denaturing buffer, allow investigating protein
complex [61]. Proteins, which were fractionized by native PAGE, form
clusters within a gel [62]. The cluster of protein complex can be subsequently separated into their individual components by denaturing
SDS-PAGE or other platform, and are identified by MS. This strategy
contribute to discovery of BAD-containing protein complex that localized to mitochondria and integrates apoptotic and glycolytic processes
[63].
The increased use of MS based identification of 2-DE separated
proteins revealed that the incidence of co-migrating proteins even in
this, the highest resolving protein method known, was more prevalent
than had been originally thought [64]. Buffers mainly affect on resolution of separated protein bands/spots. The use of alternative buffer
systems involving different carrying ions [65]. The alternative to glycine buffer, which is the most commonly used in SDS-PAGE, are tricine
[66], borate [67], bicine [68], acetate [69], asparagine, AMPSO,
HEPES, MOPS, and MES [70]. The pursuit of extreme separation of
proteome often made three-dimensional gel electrophoresis (3-DE) beyond 2-DE. In fact, general 3-DE has been used to purify particular
protein families or to investigate protein complex following native
PAGE, however, optimized a third additional step of in-gel separation to
alleviate co-migration associated drawbacks and contributed to protein
and peptide identification from MS analysis [69].
Table 1
Comparison of stain sensitivity and dynamic range of common MS compatible
technique in SDS-PAGE. Information is extracted from Gauci et al. [26].
Staining technique
Lowest limit of
detection
Linear dynamic
range
Reference
Coomassie Brilliant Blue
(CBB) G-250
CBB R-250
Colloidal CBB
Commercial colloidal CBB
Silver Nitrate
DIGE
(200–400 pmol/50 μg
protein)
SYPRO Ruby
Deep Purple
30 ng
0.5–20 μg/cm
[88–91]
10 ng
0.1–1 ng
8–16 ng
1 ng
0.25 ng
10–200 ng
30–500 ng
30–250 ng
4–50 ng
3–4 orders of
magnitude
[92]
[93,94]
[95]
[96]
[37,97]
1–2 ng
64 pg/band
[95,98–100]
[101]
Pro-Q Emerald (488)
Pro-Q Diamond
300 pg
1–2 ng
1–100 ng
4 orders of
magnitude
~9–600 ng/lane
500–100-fold
[102]
[103]
[51]. Thus, application of liquid chromatography to the analysis of
peptide mixtures generated by the proteolysis of complex protein
samples was a considerable step toward gel-independent proteomic
technologies [52]. Instead of omitting a protein gel electrophoresis step,
separating peptides directly using one or more orthogonal chromatography steps following in-solution digestion results in better protein
identification coverage (Fig. 3) [40,53]. In addition, 2-DE–MS incompatible proteins, which include low-abundance proteins such as
transcription factors, protein kinases, and other regulatory proteins, are
detectable and quantifiable in LC-ESI-MS/MS, and thus the LC-based
proteomics acquired a routine procedure for global proteome analysis
[1].
The contemporary trend of disease research has been altered to
high-throughput omics approaches which involve not only proteomics
but also genomics, transcriptomics and metabolomics. In this context, it
stands to reason that gel-free LC based mass spectrometry became a
dominant player in modern proteomics.
5. Is Gel-based proteomics still valuable?
It is not deniable that gel-free proteomics is the golden standard for
proteomics; nevertheless, gel electrophoresis is still an important
technique in disease and targeted proteome research. Why were gel
methods not disappeared? What is the comparative advantage of gel?
5.1. Gel-based proteomics as a specific fractionation method
In terms of effective fractionation methods for in-depth proteome
profiling, gel-free approach is preferred rather than gel-based approach.
Many of fractionation methods required for gel-free methods need
specific equipments such as OFFGEL fractionator and fast protein liquid
chromatography system. If a study require straightforward specific
protein enrichment rather than wide proteome coverage, gel-based
proteomics will be a good choice. Because SDS-PAGE or 2-DE provide
diverse range of separation and visualization like specific series of
phosphorylated forms of a protein, it has become a universal technique
globally used in not only proteomics core laboratories but also general
biology and disease research laboratories. Since it is easy to visualize
the gel by various staining methods as described above, and the protein
band or spot allows to qualitative assessment [71].
4. Advanced techniques evolved from gel electrophoresis
There are many attempt to overcome the weakness and to reinforce
the strength of gel-based proteomic approach. In-gel digestion is the
sine qua non of mass spectrometric protein analysis from gel electrophoresis. Gel, consisted of polyacrylamide, is not melted through digestion process that cause the difficulty of recovering the digest. One of
the tangible results is dissolvable gel. Bis-acrylcystamine (BAC)-crosslinked polyacrylamide gels, which completely dissolved by reduction,
was developed to solve recovery issue, even 40 years ago [54]. However, that technique came to a halt due to inferior resolution, timeconsuming pH adjustment, and loss of cysteine rich proteins during gel
dissolution. BAC-PAGE has been improved to efficient peptide/protein
recovery throughout complete gel dissolution [55,56]. In modern times,
BAC-PAGE not only elutes peptides/proteins efficiently, but also, has
the advantage from conventional SDS-PAGE, which particularly compatible with crude tissue extracts.
In-gel digestion has shown robustness against impurities, which
interfere with digestion or MS sensitivity, particularly detergents.
Detergents are necessary to achieve complete proteome solubilization,
however, it is hard to remove of SDS in-solution [57]. In-gel digestion
also has several limitations of using detergent, and that is mainly due to
optimal protein separation. Tube-gel digestion, which is a kind of
modified in-gel digestion, was introduced as a detergent elimination
methods [58]. In this protocol, the solubilized proteins are directly
mixed with monomeric acrylamide prior to polymerization, which allows us to use high concentrations of detergents for the solubilization of
hydrophobic proteins. Without electrophoresis, tube-gel digestion and
5.2. Specialized application of gel-based approach
Protein fractionation assisted by gel separation can be applied to
various protein-centric analysis that cannot be done by peptide-centric
fractionation (Fig. 4). Gel-based proteomics have intrinsic advantages
to LC-ESI-MS based ones.
First, the bottom-up peptide-based proteomics represented by gelfree approach can lead incorrect protein assignment due to the complexity of peptide digest. Gel-based analysis is relatively free from falsepositive issue because that handles the proteome from protein hierarchy [24].
Second, spot-targeting process is separated from protein identifying
process by mass spectrometry, and stained densitometry commonly
supports quantification process. Even though its protein co-migration
and linear dynamic range issues, densitometric measure provides more
direct quantitative results than peptide intensity based calculation.
12
BBA - Proteins and Proteomics 1867 (2019) 9–16
Y.-I. Kim, J.-Y. Cho
Fig. 3. Gel-free proteomics workflow. (a) Extracted proteins are digested without gel electrophoresis. (b) Peptide mixtures are enriched (e.g. tags, PTMs) and/or
fractionated (e.g. SCX, high-pH RP) to reduce complexity, which contribute to enhanced sensitivity on MS. (c) Peptides are separated by reverse phase liquid
chromatography, followed by ionization via nano ESI and are inducted to MS. (d) Each peptides is measured in MS spectrum, followed by selection and isolation,
subsequently fragmented to get the sequence information from MS/MS spectrum.
detection efficiency for different peptides in bottom-up is difficult to
control [75]. Moreover, intact protein analysis by top-down MS for the
2DE separated proteins can provide essential information from protein
identity to sample heterogeneity, ligand binding, substrate turnover to
structural topology, and dynamics of assembly [76,77]. Besides, the
bottle-neck of top-down approach accounted by scan range, fragmentation efficiency and resolution of MS has been improved significantly
by mass spectrometer technology [78,79]. Recently, Li et al. demonstrated the detection of protein complex up to 1.8 MDa and direct sequence determination by top-down approach [80]. We have seen that
LC-ESI-MS-based bottom-up proteomics is dominated by the improvement of liquid chromatography and MS performance and data computing. Similarly, with the improvement of MS performance, gel electrophoresis-based top-down proteomics approach will again prove
Third, gel separation can identify post-translational modification
(PTM) and protein isoforms institutively, and provide a clue for further
PTM analysis. Many proteins undergo PTM such as protein truncation,
phosphorylation, glycosylation and protein-protein ligation. These
modifications play important roles in physiological homeostatic status
or disease condition. The final product from a protein coding gene may
exist as several isoforms, which cannot be distinguished from tryptic
peptide [72]. For example, ferritine in the fruit fly exist as several
different isoforms, which localize in different pI and molecular weight
region. Two studies revealed that this protein can be analyzed by 2-DE
platform and not be in gel-free approach [73,74].
Fourth, gel electrophoresis can be a key part of top-down proteomics. Top-down MS is an alternative way to roll up the data and
complementary to bottom-up proteomics because the variation in
13
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Y.-I. Kim, J.-Y. Cho
Fig. 4. Prospects of gel-based proteomics.
by GeLC-MS provides a complete topographical description of the impact of proteolysis on protein structure [86]. It is expected that combining protease-substrate discovery platforms with ABPP should offer a
versatile and potentially routine way to map deregulated proteolytic
pathways in pathophysiological processes [87].
Despite gel-based proteomics became a minor player in recent years,
in accordance with paradigm shift to post-translational modifications,
isoform, top-down, and protein activity analysis, gel-based proteomics
will be a good complement to bridge the gap of gel-free approach and
for the detailed protein mechanism and disease mechanism studies.
important tool for targeted mechanistic and disease proteomics.
Lastly, gel-based assay is suitable platform for activity-based protein
profiling (ABPP). The measurement of enzyme activity offers both the
information of protein function and the clue of therapeutics discovery.
Functional activities of proteins are mediated by many factors, including protein-protein and protein-small-molecule interaction, and
endogenous inhibitors; thus, the protein abundance provide only an
indirect estimate of dynamics in enzyme function [81]. To measure the
enzyme activity, chemically synthesized probes, which consist of a reactive group and a reporter group, are used to profile the functional
state of enzymes directly in native proteomes [81,82]. The reporter
group of ABPP probes allows enrichment and detection of probe-modified enzymes using various methods, which include native-PAGE, gelfree proteomics, and even in vivo imaging techniques [50,83].
Even though gel-free-based ABPP provides greater proteome coverage, gel-based ABPP has some advantages compared to gel-free-based
ABPP. Gel-based ABPP is faster and more economical than gel-free
ABPP. While gel-free methods would certainly be a preferred method
for the in-depth comparison of a handful of proteomes, the analysis of
dozens or hundreds of samples will likely require gel-based approach
[84]. In addition, gel-based methods requires 0.02-fold smaller quantities of proteomes compared to gel-free methods; thus, the rare samples
such as primary human biopsies could be analyzed by fluorometric
assays on the gel [84]. Furthermore, Komatsu et al. developed ‘diced
electrophoresis gel’ assay to enhance sensitivity of fluorescent probe on
native 2-DE that complement relatively small amounts of sample on the
gel [85].
Meanwhile, gel-based approach enable proteolytic substrate to be
comprehensively characterized in endogenous settings [86]. Integrating
SDS-PAGE migratory rates with intensity of cleaved substrates acquired
Conflict of interest
The authors declare no conflicts.
Acknowledgements
This work was supported by a grant of the Bio & Medical
Technology Development Program of the National Research Foundation
(NRF) funded by the Ministry of Science and ICT, Republic of Korea
(grant number: 2016M3A9B6026771) and a grant of the Korea Health
Technology R&D Project through the Korea Health Industry
Development Institute (KHIDI), funded by the Ministry of Health &
Welfare, Republic of Korea (grant number: HI13C2098).
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