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A sensitive, robust and cost-effective approach for tyrosine phosphoproteome analysis Mingming Dong, Yangyang Bian, Yan Wang, Jing Dong, Yating Yao, Zhenzhen Deng, Hongqiang Qin, Hanfa Zou, and Mingliang Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02078 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017
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A sensitive, robust and cost-effective approach for tyrosine
2
phosphoproteome analysis
3 4 5
Mingming Dong1,2,4, Yangyang Bian1,3,4, Yan Wang1,2, Jing Dong1, Yating Yao1,2,
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Zhenzhen Deng1,2, Hongqiang Qin1, Hanfa Zou1,#, Mingliang Ye1,*
7 8
1
9
Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National
10
Academy of Sciences (CAS), Dalian 116023, China;
11
2
University of Chinese Academy of Sciences, Beijing 100049, China
12
3
Medical Research Center, The First Affiliated Hospital of Zhengzhou University,
13
Zhengzhou University, Zhengzhou, Henan 450052, China
14
4
These authors contributed equally to the work
15
*
To whom correspondence should be addressed: (M.L. Ye) Phone: +86-411-84379610.
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Fax: +86-411-84379620. E-mail:
[email protected].
17
#
Deceased April 25, 2016
18 19 20 21 22 23
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Abstract
2
Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr
3
phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction.
4
However, its analysis at the proteome level remains challenging. The conventional
5
immunopurification (IP) approach using antibodies specific to pTyr sites is known to
6
have low sensitivity, poor reproducibility and high cost. Our recent study indicated
7
that SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody
8
for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this
9
study, we presented an efficient SH2 superbinder based workflow for the sensitive
10
analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr
11
peptides than the previous method. Its excellent performance was demonstrated by the
12
analysis of a variety of different samples. For the highly tyrosine phosphorylated
13
sample, e.g. pervanadate-treated Jurkat T cells, it identified over 1800 high confident
14
pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells where the
15
pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of
16
proteins, which was 31% more than that obtained by the antibody-based method. For
17
the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5
18
mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and
19
cost-effective approach and would have wide applications in the study of the
20
regulatory role of tyrosine phosphorylation in diverse physiological and pathological
21
processes.
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Introduction
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Aberrant regulation of tyrosine phosphorylation (pTyr) often plays an important
3
role in the initiation and progression of various types of diseases, especially cancer.
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Analysis of tyrosine phosphorylation at the proteome level will shed light on the
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regulatory role of tyrosine phosphorylation in diverse physiological and pathological
6
processes. Enrichment of pTyr peptide from cell lysate digest by anti-pTyr antibody
7
followed by mass spectrometry identification, is a widely used strategy in tyrosine
8
phosphoproteomics. At present, a few site-specific anti-pTyr antibodies are
9
commercially available, i.e. P-Tyr-100, P-Tyr-1000, 4G10, and PY99. There are
10
numerous publications on immunopurification of pTyr peptides using these antibodies
11
1-6
12
decrease the sample complexity and increase pTyr enrichment selectivity 7-9. Although
13
prevailing for pTyr phosphoproteomics analysis, the antibody based pTyr enrichment
14
strategy has the drawbacks of high cost, and when used in unsaturation amount, often
15
causes the issue of poor reproducibility. For example, Palma et. al. performed 6
16
replicate pTyr immuno-affinity purification (pY-IP) experiments 4, and found the
17
number of identified pY peptides ranging from 500 to approximately 1000, with the
18
RSD as high as 42%, showed the poor reproducibility of the antibody based method.
19
SH2 domain is a sequence-specific phosphotyrosine-binding module present in many
20
signaling molecules
21
enrichment of pTyr peptides for the global tyrosine phosphoproteomics analysis
22
because its affinity to the pTyr is too low (0.1 to 10 µM)
. The combined use of immunopurification and IMAC or MOAC was reported to
10
. Wild type SH2 domains are not good for the effective
11
. By introducing three 3
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mutations into the pTyr-binding pocket of the wild type Src SH2 domain, an SH2
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superbinder, exhibiting nano-to-micromolar affinities to pTyr, was created 12. We have
3
demonstrated that this SH2 superbinder is a good replacement of pTyr antibody for
4
phosphoproteomics analysis
5
pTyr sites from 9 cell lines using this approach. However, due to the using of multiple
6
desalting steps, the enrichment procedure in that initial study is tedious and
7
cumbersome. Recently, we presented a new strategy to elute pTyr from the SH2
8
superbinder by using biotin-pYEEI as the competitive reagent
9
could be fractionated according to their binding affinities to SH2 superbinder when
10
stepwise elution with biotin-pYEEI of different concentrations was applied. However,
11
the competitive reagents used for elution must be removed before LC-MS/MS
12
analysis. This additional purification step will also result in significant sample loss.
13
The low recovery of these methods limited their applications to untreated cells or
14
tissue samples with much lower pTyr levels
13
. We have successfully identified more than 10,000
14
. The pTyr peptides
15
In this study, we presented an efficient SH2 superbinder based approach for the
16
sensitive analysis of tyrosine phosphoproteome. It combined the specific
17
phosphopeptide enrichment methods of Ti4+-IMAC with the SH2 superbinder affinity
18
chromatography in a seamless way which significantly reduced sample loss and
19
improved the robustness of the method. Compared with our previous method, this
20
new method can identify 41% more pTyr peptides. Compared with antibody 4G10,
21
this method can identify 31% more pTyr sites. The excellent performance of this
22
strategy was further demonstrated by analyzing a tissue sample of mouse skeletal 4
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muscle and epidermal growth factor (EGF) stimulated HeLa cells. For the mouse
2
muscle, we effectively identified 197 high confident pY sites (of which 73 were novel)
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from 5 mg protein digest. For the EGF stimulated HeLa cells, 262 pTyr sites were
4
quantified from 5 mg stable-isotope dimethyl labeled protein digest. Overall it is a
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sensitive, robust and cost-effective approach for tyrosine phosphoproteome analysis.
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Experimental Section
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Tyrosine Phosphorylated (pTyr) Peptide Enrichment
8
Four distinctive experimental workflows (Figure 1) were executed for the
9
enrichment of tyrosine phosphopeptides. Workflow A used SH2 superbinder
10
immobilized on Ni2+-NTA beads as affinity material, other workflows used SH2
11
superbinder covalently immobilized on CNBr-activated sepharose beads as affinity
12
material (Refer to the detail procedure in the supporting information for the
13
immobilization of SH2 superbinder). For the antibody-based method, the procedure is
14
identical to workflow C except the commercially available anti-phosphotyrosine
15
antibody 4G10 (agarose conjugate, Millipore, USA) was used as affinity material.
16
These four workflows were applied to enrich pTyr peptides from the same amount (2
17
mg) of protein digest derived from pervanadate-treated Jurkat T cells. The detailed
18
experimental procedures for the four workflows were described below.
19
Experimental workflow A was the same as used in our previous study 13. Firstly,
20
the desalted peptides were dissolved in ice-cold IAP buffer containing 50 mM
21
Tris-HCl (pH 7.2), 50 mM NaCl and 10 mM Na2HPO4. Secondly, the
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hexahistidine-tagged SH2 superbinder were purified by Ni2+-NTA beads. The 5
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Ni2+-NTA beads containing 900 µg SH2 superbinders were washed with two column
2
volumes of IAP buffer, split into three aliquots (300 µg/aliquot). Then, each aliquot
3
was incubated with 2 mg peptides at 4 °C overnight with rotating. In the next morning,
4
the beads were washed with ten column volumes of ice-cold IAP buffer, and then
5
eluted with elution buffer (500 mM imidazole in PBS buffer, pH 7.2). The eluate was
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acidified and desalted on OASIS HLB columns. The peptides eluted from the OASIS
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HLB columns were further enriched with Ti4+-IMAC beads to enhance the enrichment
8
specificity. The resulting phoshopeptides were lyophilized to dryness and dissolved in
9
0.5% FA for RPLC-MS/MS analysis.
10
For experimental workflow B, firstly, the hexahistidine-tagged SH2 superbinder
11
proteins were purified by Ni2+-NTA and covalently immobilized on CNBr-activated
12
sepharose beads. The protein concentration of the sepharose slurry was determined to
13
be 0.5 mg/mL. Secondly, the desalted peptides (2 mg) were dissolved in cold IAP
14
buffer and incubated with the SH2 superbinder (300 µg) immobilized sepharose beads
15
at 4 °C overnight with rotating. Thirdly, the sepharose beads were washed three times
16
with cold IAP buffer and two times with water. Then the bound peptides were eluted
17
twice by 0.1% TFA at room temperature. Finally, the eluate was collected and further
18
incubated with Ti4+-IMAC beads for the enrichment of phosphopeptide. The resulting
19
phoshopeptides were lyophilized to dryness and dissolved in 0.5% FA for
20
RPLC-MS/MS analysis.
21
For experimental workflow C, firstly, protein digests (2 mg) were subjected to
22
Ti4+-IMAC enrichment for phosphopeptides. Secondly, the phosphopeptides were 6
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dissolved in cold IAP buffer and incubated with sepharose beads containing
2
covalently immobilized SH2 superbinders (300 µg) at 4 °C overnight with rotating.
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The next morning, the sepharose beads were washed three times with cold IAP buffer
4
and two times with water. Finally, the bound peptides were eluted twice by 0.1% TFA
5
at room temperature. The eluate was collected and lyophilized for RPLC-MS/MS
6
analysis.
7
For experimental workflow D, firstly, the desalted peptides (2 mg) were dissolved
8
in cold IAP buffer and incubated with SH2 superbinder (300 µg) immobilized
9
sepharose beads at 4 °C overnight with rotating. Secondly, the sepharose beads were
10
washed three times with cold IAP buffer and two times with water. Finally, the bound
11
peptides were eluted twice with 0.1% TFA. The elution was lyophilized to dryness
12
and dissolved in 0.5% FA for RPLC-MS/MS analysis.
13
Workflow C was applied to enrich pTyr peptide from the mouse skeletal muscle
14
sample and EGF treated sample. The initial protein amounts were 5 mg for both
15
samples, while the amounts of immobilized SH2 superbinder used for enrichment
16
were 100 µg and 200 µg for mouse skeletal muscle samples and EGF treated samples,
17
respectively.
18
Stable Isotope Dimethyl Labeling
19
The EGF stimulated and unstimulated HeLa cells were subjected to heavy and
20
light stable isotope dimethyl labeling, separately. In brief, 2 mL of CD2O (4%, v/v) or
21
CH2O (4%, v/v) was added into 5 mg EGF stimulated or unstimulated HeLa cell
22
digests, respectively. Then 2 mL of freshly prepared NaBH3CN (0.6 M) were added 7
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subsequently to both samples. The resultant mixtures were incubated for 1 h at room
2
temperature followed by the addition of 50 µL of ammonia (25%, v/v). The mixture
3
was allowed to stand for another 15 min before formic acid (FA) was added to adjust
4
the pH to 2~3. Finally, the two heavy and light dimethyl labeling samples were
5
combined
6
phosphopeptides were lyophilized and stored at -80 °C for further usage.
7
Mass Spectrometric (MS) Analysis
together
for
phosphopeptide
enrichment
by
Ti4+-IMAC.
The
8
The nano RPLC−MS/MS experiments were performed on an UltiMate 3000
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RSLCnano systems (Thermo Scientific, USA) connected to a Q Exactive mass
10
spectrometer (Thermo Scientific, USA). For the LC-MS/MS analysis, the sample was
11
automatically loaded onto the C18 trap column (3 cm × 200 µm i.d.) at a flow rate of
12
5 µL / min with 0.1% formic acid (FA) as loading buffer. The 75 µm i.d. analytical
13
column was packed with C18 AQ particles (5 µm, 12 nm) to 15 cm length. The
14
mobile phase A was 99.9% water / 0.1% FA, and mobile phase B was 80% ACN / 0.1%
15
FA. The elution gradient executed was 5% to 35% mobile phase B lasted for 78 min.
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The Q Exactive mass spectrometer was operated in the data dependent mode.
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Survey scan MS spectra (m/z 400−2 000) were acquired by the Orbitrap with 70 000
18
resolution (m/z 200), and the AGC target was set to 1 × 106 with a max injection time
19
of 120 micro seconds. Dynamic exclusion was set to 30 s. The 12 most intense
20
multiply charged ions were fragmented by higher-energy collisional dissociation
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(HCD). The MS/MS scans were also acquired by the Orbitrap with 35 000 resolution
22
(m/z 200), and the AGC target was set to 1× 105 with a max injection time of 120 8
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micro seconds. Typical mass spectrometric settings were as follows: spray voltage, 2
2
kV; heated capillary temperature, 250 °C; normalized HCD collision energy, 27%.
3
Database Search and Data Analysis
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The raw data files generated by the Q Exactive mass spectrometer were searched 15
5
with software MaxQuant
version 1.3.0.5, against the uniprot human database
6
(released on December 11, 2013 and containing 88473 protein sequences) or the
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uniprot mouse database (released on December 11, 2013), supplemented by
8
frequently observed contaminants, and reversed versions of all sequences were
9
contained. Enzyme specificity was set to trypsin (KR/P), up to two missed cleavage
10
sites were allowed. Phospho (STY), oxidation (M), loss of ammonia and water were
11
chosen for variable modifications, carbamidomethyl was set as fixed modifications.
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The maximum false-discovery rate (FDR) was set to 1% for both the peptides and
13
proteins. The minimum required peptide length was set at six amino acids. For stable
14
isotope dimethyl labeling samples, the multiply was set as 2, Lys0 and Nter0 were
15
chosen for light label, Lys4 and Nter4 were chosen for heavy label. All the
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phosphorylation sites reported in this study were class I sites, defined by the
17
combined cutoff values of protein FDR < 1%, peptide FDR < 1%, localization
18
probability > 0.75 and ΔPTM score ≥ 5.
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Raw data repository
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All the mass spectrometry proteomics data have been deposited to the
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ProteomeXchange Consortium via the PRIDE16 partner repository with the dataset
22
identifier PXD005838. 9
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Please refer to supporting information for more experimental conditions.
2
Results and discussion
3
Establishment of the efficient SH2 superbinder based pTyr peptide enrichment
4
strategy
5
To develop a sensitive and robust affinity purification approach for tyrosine
6
phosphoproteome analysis, a seamless workflow to minimize sample loss while keep
7
high specificity should be established. To this end, we investigated the performances
8
of four different experimental workflows for the enrichment of tyrosine
9
phosphopeptides using SH2 superbinder. In the workflow A (Figure 1A), the SH2
10
superbinders were chelated on Ni2+-NTA beads via its His tag
13
11
experimental workflow has two desalting steps, which was tedious and
12
time-consuming. To simplify the experimental procedure, in this study, we covalently
13
immobilized the SH2 superbinder to CNBr-activated agarose beads, and applied them
14
in the workflow B-D (Figure 1B-D). Workflow B was similar to A, except that the
15
bound pTyr peptides were eluted by 0.1% TFA, and the second SPE desalting step
16
was omitted. As for workflow C, the Ti4+-IMAC enrichment was applied before SH2
17
superbinder enrichment. Since the protein digests with urea could be directly
18
subjected to Ti4+-IMAC enrichment, no desalting step was required. The experimental
19
workflow D aimed to test if the Ti4+-IMAC enrichment could be omitted for pTyr
20
phosphoproteomics analysis.
. The whole
21
These four workflows were applied to enrich pTyr peptides from the same
22
amount (2 mg) of protein digest derived from pervanadate-treated Jurkat T cells. 10
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Three replicates enrichments were performed for each workflow, and the obtained
2
peptides were submitted to LC-MS/MS analysis by Q Exactive mass spectrometer,
3
separately. The detailed identification results for each enrichment experiment were
4
summarized in Table S1 and S2. It should be mentioned that strict criteria was applied
5
to filter the searching results. All the pTyr peptides reported were filtered with cutoff
6
values of protein FDR < 1%, peptide FDR < 1% and score ≥ 40 ， all the
7
phosphorylation sites reported in this study were class I sites, defined by the
8
localization probability > 0.75 and ΔPTM score ≥ 5. The combined pTyr peptide
9
identifications for three replicate enrichments of each workflow were depicted in
10
Figure 2A. Obviously, the experimental workflow C yielded the highest number of
11
pTyr peptide identifications (3519), which was about 41% more identifications than
12
workflow A (2494) and B (2488), and 62% more than workflow D (2171). We also
13
compared the enrichment specificity defined as the proportion of pTyr peptides in
14
total identifications for these strategies. The workflows A, B, C have higher
15
specificity of 87.6%, 89.8% and 90.1% compared with that of 61.4% for workflow D,
16
(Figure 2A). The reason why workflow A and B identified less number of pTyr
17
peptides was attributed to that at least one SPE desalting step was employed, which
18
would lead to significant sample loss. As for workflow C, because the denaturing
19
reagent (urea) in protein digest was compatible with Ti4+-IMAC enrichment, no SPE
20
desalting was required, which effectively reduce the sample loss. The first step
21
Ti4+-IMAC enrichment can not only remove the non-phosphorylated peptides, but
22
also remove substances interfere with the next step SH2 superbinder enrichment. Also, 11
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the binding strength between phosphopeptide and Ti4+-IMAC is quite strong, which
2
minimized the sample loss. All these made workflow C an optimal strategy. On the
3
contrary, workflow D without Ti4+-IMAC enrichment step identified the lowest
4
number of pTyr peptides (2171) with the poorest specificity (61%). From Figure 2A
5
we can see that, the non-phosphopepides made a large proportion in the identification
6
results of workflow D, which indicated that many non-phosphopeptides still presented
7
in the sample which may interfere with the detection of pTyr peptides. Clearly the
8
Ti4+-IMAC purification step is very helpful for improving the specificity of pTyr
9
peptide enrichment and increasing the identification sensitivity. High specificity can
10
also be achieved by competitive elution of the captured pTyr peptides using
11
competitive reagents as we reported recently14. However, the competitive reagents
12
must be removed before LC-MS/MS analysis. This additional purification step will
13
result in sample loss and cannot compete the protocol developed in this study.
14
Reproducibility is critical for the analysis of Tyr phosphoproteome. As mentioned
15
above, to reduce the error caused by a single experiment operation, we performed
16
three replicates for each enrichment strategy. The relative standard deviation (RSD,
17
n=3) for the pTyr peptide identification numbers achieved by the workflow A, B, C, D
18
were 4%, 4%, 2% and 4% (Table S1 and Figure S1), respectively, indicating good
19
repeatability of the SH2 superbinder based enrichment. The RSD (n=3) values for the
20
ratio of pTyr peptides in workflow A, B and C were less than 1%, while for workflow
21
D, it was slightly higher, 3%. All these demonstrated that the SH2 superbinder based
22
method was highly reliable. Take the results of workflow C (the optimal method) for 12
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example, the three independent enrichment experiments C1, C2 and C3 identified
2
2545, 2702 and 2753 pTyr peptides, respectively. More than 87% of pTyr peptides
3
identified in one enrichment experiment can also be identified by the other two
4
experiments (Figure 2B), indicating good reproducibility of the method.
5
In our previous study13, we compared the enrichment performances between SH2
6
superbinder and antibody, either 4G10, P-Tyr-100 alone or an antibody mixture
7
containing ~1/3 each of 4G10, P-Tyr-100 and PY-99. We found that the SH2
8
superbinder outperformed the antibody-based methods, and between different
9
antibodies, 4G10 performed slightly better than the P-Tyr-100 or the antibody mixture.
10
In this study, we further compared the immobilized SH2 superbinder with antibody
11
4G10 for the enrichment of pTyr peptides. Instead of using pervanadate-treated
12
sample, the lysate digest derived from Jurkat cells without any stimulation was used
13
as the test sample. It is an extremely challenge sample as the pTyr peptides are present
14
at very low abundance. Equal molar amount of 4G10 and SH2 superbinder (capacity
15
equivalent to 0.6 nmol 4G10) were used to enrich pTyr peptides from 5mg protein
16
digests according to workflow C (the immobilized SH2 superbinder was replaced by
17
4G10 for the antibody based method). The obtained pTyr peptides were subjected to
18
LC-MS/MS analysis. pTyr peptides were successfully identified by both methods, but
19
the numbers were much lower than that identified from the pervanadate-treated
20
sample. The SH2 superbinder identified 481 pTyr peptides and 343 high confident
21
pTyr sites, while the 4G10 method identified 356 pTyr peptides and 262 sites (Figure
22
3A&B, Table S3). The SH2 superbinder method identified 35.1% and 30.9% more 13
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pTyr peptides and sites, respectively. We also compared the average intensity of the
2
pTyr sites identified by the two affinity reagents (Figure 3C). It shows that pTyr sites
3
presented in the SH2 superbinder dataset with an average intensity of 1.5 times higher
4
than that in 4G10 dataset, which further proved that the SH2 superbinder based
5
strategy compared favorably to antibody based approach. We then investigate the
6
binding specificity of SH2 superbinder and 4G10 to pTyr peptides under the current
7
experimental conditions. Weblogos
8
in Figure 3D. It can be seen that the amino acid sequence surrounding pTyr sites were
9
similar between these two dataset, with D, E overpresented on +1 position, E,N on +2
10
position, and L, V, I, P on +3 position. In our previous study 13, we found that different
11
affinity reagents displayed distinct specificities when used in an amount insufficient to
12
saturate pTyr peptides from the samples. And the difference in motif selectivity
13
became smaller when using more affinity reagent. This is because the affinity reagents
14
primarily enrich the pTyr peptides with stronger binding affinity when they are not
15
sufficient and so display distinct specificity, while they gradually enrich pTyr peptides
16
with weaker binding affinity when they are sufficient and so display similar
17
specificities. The high similarity of the binding specificity for SH2 superbinder and
18
4G10 in this study may because the affinity reagents used here is sufficient to saturate
19
the pTyr peptides in the sample as pTyr rarely occurred in the cells without any
20
stimulation. Above data indicated that our method outperformed the conventional
21
antibody based method and was applicable to analyze sample with extremely low
22
pTyr level.
17
were generated for each dataset and presented
14
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1
Analysis of the tyrosine phosphoproteome of mouse skeletal muscle
2
Above experiments indicated the SH2 superbinder based method is able to
3
efficiently recover pTyr peptides from the cellular lysate digest of a single type of cell
4
line. We then investigate the efficiency of this method for the analysis of a more
5
heterogeneous sample, i.e. tissue sample. Skeletal muscle has been widely studied to
6
understand the molecular basis for energy metabolism, muscle contractile function
7
and insulin resistance related signal transduction
8
phosphorylation plays an important role in modulating muscle contraction, glucose
9
transport, glycogen synthesis and protein synthesis in skeletal muscle
18-20
. It has been reported that Tyr
21
. The
10
optimized workflow was applied for enrichment of pTyr peptides from 5 mg mouse
11
skeletal muscle protein digest. The LC-MS/MS analysis of the enriched sample
12
resulted in the identification of 264 pTyr peptides and 197 high confident pTyr sites.
13
The list of all identified pTyr peptides was available at Table S4. Zhang et. al.
14
performed
15
phosphoproteome in rat skeletal muscle, which is similar to our protocol D. They
16
identified 87 pTyr sites from 10 mg protein digests, only accounted for 44.2% of the
17
number achieved in this study. Comparing the sites identified in this study with the
18
sites deposited in the PhosphoSitePlus
19
that 74 sites (37.6%) were novel. The high percentage of novel sites compared with
20
this comprehensive database (including 9140 pTyr sites on mouse proteins) indicated
21
the high sensitivity of our approach. It was known that tyrosine phosphorylation plays
22
key role in regulating glycogenolysis. After close examination of our data, we observe
one
step
P-Tyr-100
enrichment
22
to
investigate
the
20
tyrosine
(downloaded on Dec. 16, 2016), we found
15
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1
that most enzymes involved in glycogenolysis (including Pygm, Pgm1, Gpd2, Pgk1,
2
Pgam2, Ldha, Enol1, Pkm2, Ldha) were tyrosine phosphorylated, as shown in Table
3
S4. We also found the four key proteins (gene name: Tnc, Tpm, Actin, Myosin)
4
participated in muscle contraction pathway were tyrosine phosphorylated. Titin is the
5
largest protein in mammalian cells and is considered to be the regulatory node that
6
integrates myocyte signaling pathways 23. Interestingly, 53 pTyr sites were identified
7
from this giant muscle protein by our method, and more than half of them (28) were
8
identified for the first time, indicating the high sensitivity of the present method.
9
Clearly the tyrosine phosphoproteome dataset obtained by the SH2 superbinder based
10
method could shed light on the regulatory role of pTyr in tissue.
11
Quantitative analysis of pTyr events in cells after EGF stimulation
12
Quantitative analysis of the dynamic change of phosphotyrosine events in
13
response to a stimulation is of great importance in understanding the downstream
14
signal cascades. To allow for the quantification of pTyr events, we next set out to
15
incorporate the stable isotope dimethyl labeling into our sequential Ti4+-IMAC and
16
SH2 superbinder enrichment strategy. In this quantitative method, the dimethyl
17
labeling step was performed before Ti4+-IMAC enrichment. To evaluate the
18
performance of this method, it was applied to quantify the difference in pTyr events
19
before and after EGF stimulation. In detail, HeLa cells were treated with EGF for15
20
min or mock treated, then were lysed and digested with trypsin, separately. 5mg lysate
21
digest derived from untreated cells was labeled with light dimethyl, while the same
22
amount of digest derived from EGF stimulated cells was labeled with heavy dimethyl. 16
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Analytical Chemistry
1
After that, the light and heavy labeled peptides were pooled together for subsequent
2
Ti4+-IMAC enrichment. Because the reagents in the peptide mixture after dimethyl
3
labeling has no interference with Ti4+-IMAC enrichment, no additional SPE desalting
4
step was performed. The sample was further subjected to the SH2 superbinder based
5
enrichment followed by the LC-MS/MS analysis. From this analysis, we identified
6
449 pTyr peptides and 317 unique high confident pTyr sites (Table S5). Among these
7
sites, 262 sites were quantified. Compared with previous studies where typically
8
around 100 pTyr sites were quantified for such a model system
9
phosphoproteome obtained in this study was impressive. As shown in Figure 4A, a
10
global increase in tyrosine phosphorylation after EGF stimulation was observed. We
11
choose the pTyr sites with fold change >±1.5 as obvious up- or down-regulated sites,
12
a cutoff value commonly used in quantitative proteomics24-26.
2,9
, the tyrosine
13
We identified 178 up-regulated pTyr sites, while only 6 down-regulated sites due
14
to the EGF stimulation. After a closer examination of the data, we observed that most
15
pTyr events related to the EGFR signaling pathway showed a significant increase in
16
phosphorylation upon EGF stimulation. To visualize the effect of EGF stimulation,
17
the responsive phosphorylation sites identified in our study were mapped to a network
18
diagram and shown in Figure 4B. 6 tyrosine autophosphorylation sites (Y998, Y1016,
19
Y1092, Y1110, Y1172, Y1197) presented in the EGFR and 2 tyrosine
20
autophosphorylation sites (Y1139, Y1148) in ERBB2 (receptor tyrosine-protein
21
kinase erbB-2) were successfully quantified with marked increase in phosphorylation,
22
which indicated their kinase activities were enhanced. Differently, 3 tyrosine 17
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1
autophosphorylation sites (Y1185, Y1189, Y1190) in INSR (insulin receptor) were
2
also quantified but only with 1.2 times increase in phosphorylation upon EGF
3
stimulation. This could be because the pTyr on these sites were decayed after the
4
stimulation for 15 min, as we knew phosphorylation on some sites peaked as short as
5
2 min
6
reported to be directly interact with EGFR, like GAB1 (Y259, Y373, Y406, Y657,
7
Y689), PLCG1 (Y472, Y771, Y775, Y783, Y1254), SHC1 (Y349, Y350, Y428) were
8
also successfully quantified. Though EGFR pathway has been extensively studied,
9
there are still 14 novel sites presented in our dataset, compared to the human
10
phosphorylation dataset downloaded from PhosphositePlus on Dec. 16, 2016,
11
including 38 388 unique pTyr sites. For example, 4 pTyr sites (Y20, Y291, Y371,
12
Y374) on STAM2 (signal transducing adapter molecule 2) were identified. Among
13
them, Y20 presented in the VHS domain of STAM2 were reported and quantified for
14
the first time in this study. VHS domains are thought to be very important in aiding
15
membrane targeting and cargo recognition, so whether the novel Y20 site participates
16
in these processes needs further investigation. VAV3 is a guanine nucleotide exchange
17
factor that plays an important role in angiogenesis and down-regulated by EGF and
18
TGF-beta, and we identified two novel EGF up-regulated pTyr sites located in the
19
RhoGEF domain (Y367) and C1 domain (Y542) of VAV3. Although the potential
20
biological significance and function of these pTyr sites need further study, we believe
21
that our data may provide new information for biologists to explore.
22
Conclusions
27
. Tyrosine phosphorylation sites presented in signaling molecules that
18
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Analytical Chemistry
1
In this study, we presented a sensitive, robust and cost-effective approach for
2
tyrosine phosphoproteome analysis. This method combined the Ti4+-IMAC and SH2
3
superbinder affinity chromatography in a seamless way and therefore enabled the
4
efficient recovery of low abundant pTyr peptides from complex cellular lysate digest.
5
We demonstrated that this method is highly robust and is readily to analyze a variety
6
of samples. In addition to the hyper tyrosine phosphorylated sample of
7
pervanadate-treated Jurkat T cells where over 1 800 pTyr sites were identified from 2
8
mg of proteins, this method was applied to analyze the samples with low pTyr level.
9
For the unstimulated Jurkat cells where the pTyr events rarely occurred, we identified
10
481 pTyr peptides and 343 high confident pTyr sites from 5 mg proteins, which were
11
25.6% and 23.6% more than those obtained by the antibody-based method. For the
12
heterogeneous sample of tissue, we identified 264 pTyr peptides and 197 high
13
confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. By
14
combining the cost-effective stable isotopic labeling, we detected 317 high confident
15
pTyr sites and quantified 262 pTyr sites in HeLa cells after EGF stimulation using
16
only 5mg starting proteins. Most of above data are unprecedented indicating high
17
efficiency of this method. Therefore, using this method, a rather complete qualitative
18
and quantitative picture of tyrosine phosphorylation signaling events can be generated.
19
It is readily applicable to analyze either cell or tissue samples to reveal the regulatory
20
role of tyrosine phosphorylation in diverse physiological and pathological processes.
21
Acknowledgments
22
This work was supported, in part, by funds from the China State Key Basic 19
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Page 20 of 28
1
Research Program Grants (2016YFA0501402, 2013CB911202), the National Natural
2
Science Foundation of China (21605140, 21235006, 21535008, 81600046). MY is a
3
recipient of the National Science Fund of China for Distinguished Young Scholars
4
(21525524). We also thank Prof. Shawn Li in University of Western (Canada) for
5
providing the SH2 superbinder plasmids.
6
Competing financial interests: The authors declare no competing financial
7
interest.
8
Supporting information available
9 10
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
11 12
References
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Figure legends
2
Figure 1. Four different experiment workflows for pTyr peptide enrichment
3
using SH2 superbinder. SH2 superbinders non-covalently chelated on Ni2+-NTA
4
beads were used in workflow A, while SH2 superbinders covalently immobilized to
5
CNBr-activated agarose beads were used in workflow B-D.
6
Figure 2. Comparison of the pTyr peptide enrichment performance for different
7
experiment workflows. (A) The numbers of identified peptides and the pTyr peptide
8
enrichment specificity for the four different experiment workflows were shown in
9
Figure 1. (B) The overlap of pTyr peptides identified by the three replicates
10
enrichment experiments of workflow C.
11
Figure 3. Comparison of the antibody 4G10 and SH2 superbinder based
12
strategies. Equal molar amount of 4G10 and SH2 superbinder (capacity equivalent to
13
0.6 nmol 4G10) were used to enrich pTyr peptides from 5mg untreated Jurkat celluar
14
protein digests according to workflow C (the immobilized SH2 superbinder was
15
replaced by 4G10 for the antibody based method). The overlap of the identified pTyr
16
peptides and pTyr sites of these two method were shown in (A) and (B), respectively.
17
(C) Comparison of the log2 intensity of pTyr sites identified by the SH2 superbinder
18
method and the 4G10 antibody method. The bottom and top edges of the box
19
represent the first and third quartiles, and the band inside the box corresponds to the
20
median of the data. The median 25.4 and 24.8 corresponding to pY intensities of
21
4.42E7 and 2.92E7, respectively. So the average intensity of pY sites in the SH2
22
superbinder dataset was 1.5 times of that in the 4G10 antibody dataset. (D) Weblogos 22
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Analytical Chemistry
1
were generated for the pTyr sites identified by the SH2 superbinder and 4G10
2
antibody based method, respectively, for the comparison of pTyr motif selectivities of
3
the two affinity reagents.
4
Figure 4. Quantitative analysis of pTyr events in cells stimulated by EGF. The
5
same amount of protein digest (5 mg) derived from HeLa cells treated by EGF for 0
6
min and 15 min were labeled with light and heavy dimethyl, separately. Then they
7
were pooled together for pTyr peptide enrichment according to workflow C. (A) The
8
Log2 values of the H/L ratios for the identified pTyr sites. (B) The responsive
9
phosphorylation sites identified in our study towards EGF stimulation were mapped to
10
a signaling network diagram. The site of phosphorylation is indicated by “Y”
11
followed by the amino acid number in the protein sequence.
12 13 14 15 16 17 18 19
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Figure 1.
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Figure 2.
2
3 4 5 6 7 8 25
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Figure 3.
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Figure 4.
2 3
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