High-Throughput Proteomics Enabled by a Photocleavable Surfactant

Article abstract of DOI:10.1002/anie.201915374

Mass spectrometry (MS)-based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high-throughput proteomics. Herein, we report a high-throughput bottom-up proteomic method enabled by a newly developed MS-compatible photocleavable surfactant, 4-hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS-analysis following UV degradation. Moreover, we developed an Azo-aided bottom-up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an “all-in-one” MS-compatible surfactant for both top-down and bottom-up proteomics, with streamlined workflows for high-throughput proteomics amenable to clinical applications.

Full text of DOI:10.1002/anie.201915374

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Accepted Article
Title: High-throughput Proteomics Enabled by a Photocleavable
Surfactant
Authors: Kyle Brown, Trisha TUCHOLSKI, Christian Eken, Samantha
Knott, Yanlong Zhu, Song Jin, and Ying Ge
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201915374
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High-throughput Proteomics Enabled by a Photocleavable
Surfactant
Kyle A. Brown, Trisha Tucholski, Christian Eken, Samantha Knott, Yanlong Zhu, Song Jin*, Ying Ge*
Abstract: Mass spectrometry (MS)-based proteomics provides
unprecedented opportunities for understanding the structure and
function of proteins in complex biological systems; however, protein
solubility and sample preparation before MS analysis remain a
bottleneck preventing high-throughput proteomics. Herein, we report
for the first time a high-throughput bottom-up proteomics method
up proteomics.[9-12]
detergents) are added to effectively solubilize proteins, especially
Typically, surfactants (also known as
membrane proteins, in cells and tissues.[3,
However,
8, 11, 13, 14]
conventional surfactants that can solubilize cells and tissue well,
such as sodium dodecyl sulfate (SDS), are incompatible with MS
and need be removed before MS analysis, which is not only time
consuming but may result in protein loss and degradation.[13, 15, 16]
An MS-compatible surfactant that can solubilize and denature
proteins with comparable efficacy to SDS without the need for time-
consuming sample clean-up is attractive for streamlining sample
preparation for proteomics.[12] To address this problem, MS-
compatible surfactants that can be degraded into innocuous non-
enabled by
a newly developed MS-compatible photocleavable
surfactant, 4-hexylphenylazosulfonate (Azo)[1] that facilitates robust
protein extraction, rapid enzymatic digestion (30 min compared to
overnight), and subsequent MS-analysis following UV degradation.
Moreover, we developed an Azo-aided bottom-up method for efficient
analysis of integral membrane proteins, which are key drug targets
and are generally underrepresented in global proteomic studies.
Furthermore, we demonstrated the unique ability of Azo to serve as
an “all-in-one” MS-compatible surfactant for both top-down and
bottom-up proteomics, with streamlined workflows for high-throughput
proteomics amenable to clinical applications.
surfactant by-products have been developed.[15, 17-19] Particularly,
20]
acid-labile ionic surfactants such as RapiGest™[18,
,
ProteaseMax™[19], and MaSDeS[17] can significantly improve the
protein solubility and facilitate protein digestion. However, these
acid-labile surfactants are degraded in highly acidic conditions post-
digestion and often require an offline clean-up step, precluding
automation.[15] Moreover, strong acidic degradation may cause the
loss of important post-translational modifications that are unstable
in acidic conditions (i.e. acid-labile phosphorylations[21] and
glycosylations[22]). Furthermore, some of these acid-labile
surfactants generate amine products following degradation that
interfere with the isobaric amine-reactive tandem mass tag labeling
reagents, such as tandem mass tag (TMT) and isobaric tags for
relative and absolute quantitation (iTRAQ).[15] Most importantly,
none of these acid-labile surfactants are compatible with top-down
proteomics for the direct analysis of intact proteins, because they
greatly suppressed intact protein MS signal as reported previously.[1]
Therefore, we aim to develop a high-throughput sample preparation
method that is amenable to both top-down and bottom-up
proteomics and overcome the limitations of the conventional acid
cleavable surfactants.
Mass spectrometry (MS)-based proteomics allows large-scale,
quantitative measurements of proteins, and provides unprecedented
insights into the structure and function of proteins in complex
biological systems.[2, 3] Currently, there are two major MS-based
proteomic approaches: the bottom-up approach, where proteins are
enzymatically digested and the resulting peptides measured by MS
to infer the identity, quantity, and modification status of the proteins;
and the top-down approach, where intact proteins are measured and
their sequences determined for complete characterization of their
proteoforms.[4-7] Bottom-up proteomics remains the most
widespread proteomic method due to the well-established
experimental and computational tools[8] developed over the last two
decades, but it has limitations for comprehensive analysis of
proteoforms.[5, 7] On the other hand, significant strides have recently
been made to advance top-down proteomics, which provides
comprehensive analysis of proteoforms, owing to the recent
successful developments of high-resolution mass spectrometers,
protein separations, and user-friendly software for data analysis.[5, 6,
Recently, we developed a photo-cleavable surfactant, 4-
hexylphenylazosulfonate (Azo), for top-down proteomics.[1] Azo is
straightforward to synthesize (requiring only two-step synthesis and
simple purification),[23] effectively solubilizes proteins (including
membrane proteins), and can be rapidly degraded before MS
9]
However, further method developments are urgently needed to
increase the throughput in both bottom-up and top-down
approaches, especially for time-sensitive clinical applications.
Sample preparation remains a bottleneck for obtaining high-quality
MS data in a high-throughput fashion for both top-down and bottom-
[a]
[b]
K. A. Brown, T. Tucholski, C. Eken, S. Knott, Prof. S. Jin*, Prof. Y.
Ge*
Department of Chemistry, University of Wisconsin-Madison
Madison, WI 53706 (USA)
E-mail: jin@chem.wisc.edu
Y. Zhu, Prof. Y. Ge
Department of Cell and Regenerative Biology, University of
Wisconsin-Madison
Madison, WI 53706 (USA)
E-mail: ge2@wisc.edu
Supporting information for this article is given via a link at the end
of the document.
Scheme 1. Scheme for azo-enable high-throughput top-down and bottom-
up proteomics.
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protein band was observed in the presence of 0.1% and 0.2% Azo
after 0.5 h of digestion indicating that Azo greatly improved
digestion efficiency. Subsequently, we evaluated a longer digestion
time (1 h) in the presence or absence of Azo. Even after 1 h
digestion, a predominant intact protein was still observed without
Azo, whereas only a minute amount of intact protein was visible in
the presence of 0.05% Azo and no intact protein was detected for
0.1% or 0.2% Azo (Figure 1ef). This is conceivable, since without
a denaturing agent (e.g. an anionic surfactant) the enzyme has
limited access to the protein backbone, precluding digestion. These
results indicate Azo facilitates denaturation of the protein, providing
efficient digestion. Furthermore, we analyzed the Azo-aided
myoglobin digest (0.1% Azo, 1 h) via direct electrospray ionization
for peptide mass fingerprinting analysis without an additional clean-
up procedure and observed 89% sequence coverage (Figure S3,
bottom panel). On the other hand, analysis of the myoglobin sample
digested in the absence of surfactant (0% Azo, 1 h) yielded minimal
detectable peptides, and the MS signal was largely dominated by the
intact protein (Figure S3, top panel). Next, we performed a direct
comparison between Azo and RG for trypsin digestion of myoglobin
and direct-infusion MS analysis, observing lower peptide signal for
the sample with RG than that with Azo (Figure S4). Finally, to
further confirm the rapid rate of enzymatic digestion in the presence
of Azo, we digested a standard mixture of insulin, myoglobin, and
carbonic anhydrase with (0.1% or 0.2%) and without Azo. We
observed a similar improvement in digestion with Azo (Figure S5).
Taken together, these results indicate proteins (even those resistant
to trypsin digestion) can be rapidly and efficiently digested using
Azo.
Figure 1. Enhanced enzymatic digestion and MS analysis using a
photocleavable surfactant, Azo. (a) Scheme for Azo-aided bottom-up
proteomics. (b) Degradation of Azo by UV irradiation. (c) UV-Vis spectrum
for Azo highlighting maximal absorbance at 305 nm. (d) UV-Vis spectrum
monitoring the rapid degradation of Azo (0.1% in water) as a function of
irradiation time with a 100 W mercury lamp. (d) Digestion of myoglobin [0-
1 h, Azo (0-0.2%)] was monitored by SDS-PAGE, stained with Coomassie
Blue, and (f) LC-MS analysis using a Q-TOF mass spectrometer. Relative
abundances of the extracted ion chromatograms was normalized to 5.5 E8.
After demonstrating rapid enzymatic digestion using standard
proteins, we further investigated Azo-aided digestion of complex
cell lysates. A single-step Azo protein extraction was performed
using human embryonic kidney 293T (HEK293T) cells to generate
whole cell lysate. SDS-PAGE analysis demonstrated reproducible
extractions for three HEK293T samples (Figure 2a). The proteins
analysis.[1] Here, for the first time, we demonstrate that Azo is fully
compatible with bottom-up proteomics and uniquely serves as an
“all-in-one” MS-compatible surfactant for both bottom-up and top-
down proteomics, which greatly facilitates high-throughput sample
handling before MS-analysis (Scheme 1). A direct comparison of
the Azo and a leading acid-labile surfactant, RapiGestTM (RG, also
known as ALS),[18, 20] clearly demonstrated Azo is compatible with
direct-infusion electrospray MS analysis of intact proteins and
peptides whereas RG greatly suppressed MS signal (Figure S1).
Moreover, we demonstrate the ability of Azo to facilitate protein
extraction, enable rapid enzymatic digestion, and rapidly degrade by
UV before MS analysis for a streamlined bottom-up proteomics
approach (Figure 1a).
First, we demonstrated the rapid degradation of Azo by UV-
Vis spectroscopy. The intact surfactant has a maximal absorbance at
305 nm[1, 24] (Figure 1b-c); thus, degradation of Azo can be achieved
using a mercury lamp (305 nm) in less than 5 min [1, 25] (Figure 1d
and S2). This facile surfactant degradation method makes Azo ideal
for rapid sample processing.
Next, we evaluated Azo-aided in-solution digestion.
Myoglobin, a trypsin resistant protein,[18] was digested in-solution
without (0%, control) or in the presence of Azo (0.05%, 0.1%, or
0.2%). The efficiency of digestion was assessed by visualization of
the remaining intact myoglobin through SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis (Figure 1e). After 0.5 h of
trypsin digestion in the absence of surfactant, the intact protein band
at ~17 kDa was clearly observed, suggesting poor digestion
efficiency. In contrast, only a faint intact protein band was observed
for sample digested in the presence of 0.05% Azo, and no intact
Figure 2. Rapid and reproducible extraction, enzymatic digestion, and LC-
MS/MS analysis of proteins from human embryonic kidney 293T
(HEK293T) cells (a) SDS-PAGE analysis demonstrates consistent
extraction profiles across three extractions (marker (M), 1, 2, 3). Aliquots
were taken from each exaction and digested overnight or for 1 h. After LC-
MS/MS and MaxQuant identification, a high degree of overlap was
observed for both the (b) overnight and (c) 1 h samples. Similarly (d) 1403
of the 1726 combined identified protein groups were observed in both
digestion methods. Data collected using Q-TOF.
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were digested for 1 h or overnight to determine whether digestion
time, the most time-consuming step in the bottom-up proteomics
workflow, could be shortened significantly using Azo while
maintaining reproducibility. After surfactant degradation by UV, a
brief centrifugation step, and liquid chromatography (LC)-MS/MS
[26]
analysis, the data was processed using MaxQuant
to determine
the number of protein groups identified in each sample and to
perform label-free quantitation (Table S1). Overall, 910 and 938
proteins were identified in all three extraction replicates using
overnight digestion (Figure 2b) and 1 h digestion (Figure 2c),
respectively. Combined, we identified 1557 proteins using an
overnight digestion and 1572 proteins using a 1 h digestion with a
60 min LC gradient before MS analysis. This corresponds to an 81%
overlap between the two conditions (Figure 2d), confirming 1 h is
sufficient for effective protein digestion in the presence of Azo.
Next, we compared the performance of Azo-aided digestion (30 min
and overnight) with a gold-standard protocol for bottom-up
proteomics using SDS, Filter Assisted Sample Preparation (FASP)
developed by Mann and co-workers (overnight digestion).[11] Using
three digestion replicates (proteins were extracted with Azo or SDS
respectively) and a 60 min LC-MS analysis, we observed the
greatest number of identifications using the Azo digestion
(overnight) followed by the FASP (overnight) and Azo digestion (30
min) method (Figure S6 and Table S2). Significantly, we observed
only 33 fewer protein identifications using a 30 min digestion
protocol with Azo when compared to FASP, demonstrating a very
robust, reproducible method that greatly reduces the sample
preparation time by eliminating the multiple centrifugation and
offline desalting steps (SI Discussion)
Figure 4. Azo-enabled top-down proteomics analysis of cardiac tissue. (a-
d) Intact mass spectra of proteoforms analyzed by LC-MS. Data collected
using Maxis II Q-TOF.
Next, we demonstrate Azo’s ability to aid in the analysis of
integral membrane proteins, which are hydrophobic and generally
expressed at lower levels, thus are underrepresented in broad
proteomics studies.[15, 27] Here, we focused on improving the bottom-
up workflow for membrane proteomics by implementing a cloud-
point extraction, using Triton X-114, to enrich membrane proteins
from HEK293T cells with a single extraction (SI Discussion).[28]
However, Triton surfactants are highly incompatible with MS
analysis[29] and require removal before MS analysis. Subsequently
we used Azo to solubilize the precipitated protein pellets enriched
with integral membranes for a more effective analysis of this
important class of proteins. We observed more than a 6-fold increase
in integral membrane protein identified using this method when
compared to whole cell extraction experiment (conceivably because
highly abundant proteins that traditionally suppress the detection of
integral membrane proteins were depleted) (Figure 3a and Table
S3). Similarly, a 4-fold increase in protein groups identified was
achieved using 30 min digestions times for the enriched sample
compared to the whole cell lysate (Figure 3a and Table S3).
Importantly, we detected several proteins with over 10
transmembrane domains, demonstrating the ability of Azo to
solubilize highly hydrophobic proteins and enable their MS analysis
(Figure 3b). The results indicate longer digestion improves the
identification of integral membrane proteins even with Azo, which
can be rationalized by the lower abundance of lysine and arginine
residue particularly in the transmembrane regions.[27] Finally, we
performed a global PTM discovery search using MetaMorephus[30]
to localize modification on membrane proteins (Figure S7 and
Table S3).
Here, we sought to demonstrate the unique application of Azo
as an “all-in-one” MS-compatible surfactant for both top-down and
bottom-up proteomics. Proteins were extracted, using Azo, from left
ventricle heart tissue (after aqueous buffer extractions) and an
aliquot was taken for bottom-up analysis. We observed that many of
the protein identifications were involved in cellular and metabolic
processes or localization (Figure S8a and Table S4). Moreover, we
demonstrated the potential of this method to investigate disease-
relevant proteins (e.g. sarcomeric proteins) and their interacting
partners[31] which is critical for gaining a better understanding of
Figure 3. Analysis of integral membrane protein enabled by Azo. Proteins
were extracted and membrane proteins enriched using a Triton x-114
cloud point extraction procedure,28 then precipitated, and solubilized again
in Azo for in-solution digestion. Additionally, Azo was used for whole cell
protein extraction. Using an (a) overnight digestion a significantly higher
number of integral membrane proteins were identified using the phase
separation enrichment followed by Azo-aided digestion. Similarly a 30 min
digestion was performed demonstrating a high-throughput membrane
protein analysis methods. (b) A table of representative membrane proteins
with a high number of transmembrane domains that were solubilized using
Azo. Data represent a single LC-MS experiment.
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their role in disease mechanisms (Figure S8b). Furthermore, an
aliquot was taken for top-down proteomics which provides
additional information about existing proteoforms (Figure 4 and
Figure S9), thus improving the biological information that can be
obtained from a sample. Overall LC-MS analysis of cardiac tissue
illustrated the complexity of the Azo-extracted samples with many
proteins and their respective proteoforms eluting simultaneously
(Figure S10), demonstrating the promise of Azo for future
applications in global top-down proteomics studies. Importantly,
Azo also enables the extraction and analysis of intact integral
membrane proteins (Figure S11).
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In summary, we developed a novel approach using an anionic
photocleavable surfactant, Azo, to enable high-throughput bottom-
up proteomics. Azo can effectively extract and solubilize proteins
reproducibly, enables rapid enzymatic digestion, and is amenable to
MS analysis without an additional desalting step which improves the
throughput, permitting bottom-up analyses for a wide range of
applications. Moreover, we have developed an Azo-aided bottom-
up proteomics workflow for effective membrane protein
solubilization. Furthermore, we have demonstrated the unique
capability of Azo as an “all-in-one” MS-compatible surfactant for
both top-down and bottom-up proteomics with great potential to
provide a streamlined strategy for high throughput proteomics with
far-reaching applications to fully realize the impact of proteomics in
clinical diagnosis.
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Acknowledgments
The financial support was provided by the NIH R01 grant
GM117058 (to S.J. and Y. G.). Y.G. also would like to acknowledge
NIH grants GM125085, HL109810, and HL096971 and the high-
end instrument grant S10OD018475 (to Y.G.). K.A.B. would like to
acknowledge support from the Training Program in Translational
Cardiovascular Science, T32 HL007936-19. T.T. was supported by
the NIH Chemistry Biology Interface Training Program,
T32GM008505. S.K. was supported by the NSF Graduate Research
Fellowship Program under Grant No. DGE-1747503 and
WiscAMP-DB program under grant No. HRD-1612530. We also
would like to thank Dr. Rosa Viner for the helpful discussions.
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Entry for the Table of Contents
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Kyle A. Brown, Trisha Tucholski, Christian Eken, Samantha Knott,
Yanglong Zhu, Song Jin*, and Ying Ge*
Page No. – Page No.
High-throughput Proteomics Enabled by a Photocleavable
Surfactant.
Photocleavable Surfactant. In their Communication,
Ge, Jin, and co-workers demonstrated the unique
ability of 4-hexylphenylazosulfonate (Azo) to serve as
an “all-in-one” MS-compatible surfactant for both high-
throughput bottom-up and top-down proteomics.
This article is protected by copyright. All rights reserved.