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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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Analytical Chemistry

1

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,

6

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.

16

Fax: +86-411-84379620. E-mail: [email protected].

17

#

Deceased April 25, 2016

18 19 20 21 22 23

1

ACS Paragon Plus Environment


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Page 2 of 28

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.

22 2


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1

Introduction

2

Aberrant regulation of tyrosine phosphorylation (pTyr) often plays an important

3

role in the initiation and progression of various types of diseases, especially cancer.

4

Analysis of tyrosine phosphorylation at the proteome level will shed light on the

5

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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1

mutations into the pTyr-binding pocket of the wild type Src SH2 domain, an SH2

2

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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1

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)

3

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

5

sensitive, robust and cost-effective approach for tyrosine phosphoproteome analysis.

6

Experimental Section

7

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

22

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

6

acidified and desalted on OASIS HLB columns. The peptides eluted from the OASIS

7

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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1

dissolved in cold IAP buffer and incubated with sepharose beads containing

2

covalently immobilized SH2 superbinders (300 µg) at 4 °C overnight with rotating.

3

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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1

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

9

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.

16

The Q Exactive mass spectrometer was operated in the data dependent mode.

17

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

21

(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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1

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

4

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

7

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.

12

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

16

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.

19

Raw data repository

20

All the mass spectrometry proteomics data have been deposited to the

21

ProteomeXchange Consortium via the PRIDE16 partner repository with the dataset

22

identifier PXD005838. 9



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Page 10 of 28

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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1

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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Page 12 of 28

1

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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1

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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Page 14 of 28

1

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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Page 16 of 28

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


Page 17 of 28

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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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Page 18 of 28

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


Page 19 of 28

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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

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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Page 22 of 28

1

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


Page 23 of 28

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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

23



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Page 24 of 28

Figure 1.

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24


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1

Figure 2.

2

3 4 5 6 7 8 25



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Page 26 of 28

Figure 3.

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1

Figure 4.

2 3

27



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