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A biphasic affinity chromatographic approach for deep tyrosine phosphoproteome analysis Zhenzhen Deng, Mingming Dong, Yan Wang, Jing Dong, Shawn S.C. Li, Hanfa Zou, and Mingliang Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04288 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Analytical Chemistry

2

A biphasic affinity chromatographic approach for deep tyrosine phosphoproteome analysis

3

Zhenzhen Deng1,2, Mingming Dong1, Yan Wang1,2, Jing Dong1, Shawn S.-C. Li3,

4

Hanfa Zou1,# , Mingliang Ye1,*

1

5 6

7

8 9

1

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China 2

Graduate School of Chinese Academy of Sciences, Beijing 100049, China

3

Departments of Biochemistry, Oncology and the Children’s Health Research

Institute, Schulich School of Medicine and Dentistry, Western University, London,

10

Ontario N6A 5C1, Canada

11

* To whom correspondence should be addressed: (M.L. Ye) Phone: +86-411-84379620. Fax: +86-411-84379620.

12

E-mail: [email protected].

13

# Deceased April 25, 2016

14 15 16

1 ACS Paragon Plus Environment


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Abstract:

2

Tyrosine phosphorylation (pTyr) is important for normal physiology and

3

implicated in many human diseases, particularly cancer. Identification of pTyr sites is

4

critical to dissecting signaling pathways and understanding disease pathologies.

5

However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis

6

of pTyr at the proteome level is more challenging due to its low abundance. Here, we

7

developed a biphasic affinity chromatographic approach where Src SH2 superbinder

8

was

9

phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI,

10

this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity

11

ones, while the excess competitive agent is readily removed by using NeutrAvidin

12

agarose resin in an integrated tip system. The excellent performance of this system

13

was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which

14

3,480 unique pTyr sites were identified. The biphasic affinity chromatography method

15

for deep Tyr phosphoproteome analysis is rapid, sensitive, robust and cost-effective. It

16

is widely applicable to the global analysis of the tyrosine phosphoproteome associated

17

with tyrosine kinase signal transduction.

coupled

with

NeutrAvidin

affinity

chromatography,

for

tyrosine

18 19


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1

INTRODUCTION

2

Protein tyrosine phosphorylation (pTyr) is of central importance in signaling

3

systems in eukaryotic cells as it regulates many important biological events including

4

proliferation, adhesion, differentiation, hormone responses and immune defense.1-3

5

The deregulation of tyrosine phosphorylation has been implicated in human diseases

6

including cancer. Tyrosine kinases, the enzymes catalyzing the phosphorylation of

7

tyrosine residues, are a major class of drug targets, and a number of tyrosine kinase

8

inhibitors have been approved for clinical use.4-6 Deep Tyr phosphoproteome holds

9

great promise for understanding tyrosine phosphorylation-regulated aberrant signal

10

transduction in human disease. Sensitive methods are highly required for the detection

11

of tyrosine phosphorylation events. Mass spectrometry (MS) has been proved to be an

12

ideal platform for the global identification of proteins and their post-translational

13

modifications (PTMs).7 Because of the low abundance of tyrosine phosphorylated

14

proteins, the coverage of Tyr phosphoproteome depends on the performance of the

15

pTyr peptide enrichment prior to MS analysis. Typically, pTyr peptides are enriched

16

from protein digest by pan-specific antibodies for pTyr, such as 4G10, P-Tyr-100 and

17

PY-99,8 for tyrosine phosphoproteome analysis. However, the high cost of these

18

antibodies prohibits their use in a sufficient amount to saturate the pTyr peptides in a

19

sample, resulting in a relatively low coverage of the Tyr phosphoproteome.9,10 Despite

20

the large number of Tyr kinases in the cell, tyrosine phosphorylation is tightly

21

regulated and maintained at low abundance (accounting for 10,000 pTyr

12

sites from 9 human cell lines, achieving unprecedented deep coverage for tyrosine

13

phosphoproteome.10

14

The SH2 domain is a sequence-specific phosphotyrosine-binding module

15

presented in many signaling molecules. There are 120 SH2 domains encoded by the

16

human genome, and each SH2 domain binds a unique spectrum of tyrosine

17

phosphorylated sites.12,13 Because SH2 domains are what the cell uses to respond to

18

changes in tyrosine phosphorylation during signaling, the binding pTyr sites of

19

different SH2 domains can provide a wealth of information about the mechanisms and

20

status of pTyr signaling. With the markedly enhanced binding affinity, almost all pTyr

21

peptides could be bound to the SH2 superbinder. Nevertheless, LC-MS/MS analysis

22

following one-step elution of all bound peptides by TFA would result in the loss of


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1

information in binding specificity and affinity for the identified pTyr sites. It’s known

2

that the SH2 superbinder and the wild-type SH2 domain have similar specificity

3

toward pTyr peptides despite large differences in affinity.10 We hypothesize that the

4

bound pTyr peptides could be sequentially eluted on basis of their binding affinity.

5

Because progressive elution of the bound pTyr peptides based on affinity would

6

provide another dimension of separation prior to LC-MS/MS analysis, it could lead to

7

deeper and broader coverage of the Tyr phosphoproteome. The superbinder used in

8

this study is derived from Src SH2 domains. pTyr-Glu-Glu-Ile (pYEEI) was found to

9

have highest affinity to Src SH2 domains by screening of degenerate phosphopeptide

10

library.14-16 Therefore, we utilized a biotinylated pTyr peptide with the sequence of

11

biotin-pYEEI as a competitive elution agent. To prevent the interference of the

12

competing reagent on the detection of the enriched pTyr peptides, a biphasic affinity

13

chromatographic approach was developed to enrich and fractionate pTyr peptides. It

14

was found that the pTyr peptides were successively released in accordance with their

15

binding affinity to the superbinder SH2 domain. A total of 7,227 pTyr peptides and

16

3,480 unique pTyr sites were identified by the sequential elution for affinity

17

purification from only 2 mg pervanadate-treated Jurkat cell lysate digest.

18

EXPERIMENTAL SECTION

19

Chemicals and Materials

20

Formic acid (FA) was obtained from Fluka (Buches, Germany). Acetonitrile

21

(ACN, HPLC-grade) was purchased from Merck (Darmstadt, Germany). NeutrAvidin

22

agarose resin was obtained from Thermo Fisher Scientific (Massachusetts, USA).



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CNBr-activated Sepharose 4B was purchased from GE (New Jersey, USA). Daisogel

2

ODS-AQ (5 μm) was purchased from DAISO Chemical CO., Ltd (Osaka, Japan).

3

Biotin-pYEEI was synthesized from ChinaPeptides (Shanghai, China). Fused-silica

4

capillaries with 200 μm and 75 μm i.d. were purchased from Polymicro Technologies

5

(Phoenix, AZ, USA). All the water in the experiments was purified by a Milli-Q

6

system from Millipore Company (Bedford, MA, USA). All other chemicals were

7

purchased from Sigma-Aldrich (St. Louis, MO, USA).

8

Expression, purification and immobilization of the Src superbinder

9

We performed the expression and purification of Src superbinder according to

10

Kaneko et al.11 The Src superbinder SH2 domain was immobilized on CNBr-activated

11

Sepharose 4B following manufacture’s protocol (GE Healthcare), and the final

12

concentration for the immobilized Src superbinder was 1 mg protein per mL medium.

13

Cell culture, protein extraction and protein digestion

14

Jurkat cells were cultured in RPMI 1640 medium with 10 % neonatal bovine

15

serum, 100 units/mL penicillin/streptomycin in a humidified atmosphere of 5 % CO2

16

at 37°C. The cells were collected by centrifugation, washed three times with PBS, and

17

treated with 1 mM pervanadate (freshly prepared by mixing 1 mM orthovanadate with

18

1 mM hydrogen peroxide) in tube for 15 min at 37°C. The procedures for cell lysis

19

and trypsin digestion were performed according to Humphrey et al.17

20

Phosphotyrosyl (pTyr) peptide enrichment

21

The protein digest was subjected to enrichment with immobilized titanium (IV)

22

ion affinity chromatography (Ti4+-IMAC) for phosphopeptides as previous reported.18


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1

The enriched phosphopeptides from 2 mg initial cell lysate were then dissolved in

2

ice-cold immunoaffinity purification (IAP) buffer containing 50 mM MOPS-NaOH

3

(pH 7.2), 10 mM Na2HPO4 and 50 mM NaCl. The sample was incubated with

4

immobilized Src superbinder (300 µg) at 4°C overnight with rotation. For the

5

application of Src superbinder-based sequential competitive elution strategy (as

6

shown in Figure 1), a sieve plate with 2 mm diameter was packed into a tip (1 mL),

7

washed with 80 % ACN containing 0.1 % TFA and 50 mM NH4HCO3 buffer (pH7.2),

8

respectively. Then, 500 µL NeutrAvidin agarose resin was packed into the tip and

9

washed with 50 mM NH4HCO3 buffer (the binding capacity was ~ 40 nmol

10

biotin-pYEEI per milliliter of resin, according to the specification of NeutrAvidin

11

resins that the binding capacity was 10 µg biotin per milliliter). Another sieve plate

12

with 5 mm diameter was packed into the tip above the NeutrAvidin. Finally, the

13

immobilized Src superbinder Sepharose beads with the captured pTyr peptides were

14

packed into the tip. This biphasic tip was washed five times with 400 µL NH4HCO3

15

buffer, and sequentially eluted with 4 µM, 8 µM, 16 µM and 32 µM competitive agent

16

(biotin-pYEEI) in NH4HCO3 buffer (300 µL). All the eluents were collected,

17

lyophilized, and stored at -30°C for further analysis.

18

For the single step competitive elution strategy, all the steps were identical with

19

these mentioned above, except the elution step using 30 µM competitive agent (400

20

µL). Another single step competitive elution strategy was performed similarly except

21

the use of NeutrAvidin. For the trifluoroacetic acid (TFA) elution strategy, the

22

NeutrAvidin agarose resin was absent, and the elution step was performed using 0.2%



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Page 8 of 23

1

TFA (400 µL). For comparison, the experiments using samples without subject to

2

Ti4+-IMAC enrichment were also performed. These samples were desalted by OASIS

3

HLB column, then incubated with immobilized Src superbinder. After washing with

4

50 mM NH4HCO3 buffer, the bound peptides were finally eluted with either 30 µM

5

biotin-pYEEI or 0.2 % TFA.

6

MS analysis, database searching and data analysis

7

Peptides were loaded onto a 14 cm-capillary column with a 75 µm inner diameter,

8

packed in-house with C18 particles (3 µm, 120 Å). For the RPLC separation, 0.1% FA

9

in water and in 80% acetonitrile were used as buffer A and B. Peptides were eluted

10

with a gradient of 4% - 35% buffer B over 85 minutes followed by 35% - 45% buffer

11

B over 10 min, resulting in approximately 95 min gradients, at a flow rate of 0.3

12

µL/min.

13

Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific), with one full

14

scan (400-2,000 m/z, R=70,000 at 200 m/z) at a target of 1e6 ions with max IT of 120

15

ms. And the ions were fragmented by higher-energy collisional dissociation (NCE

16

28%), followed by ten data-dependent MS/MS scans with a resolution of 35,000 at

17

200 m/z (target 5e5 ions with max IT of 100 ms, isolation window 2.0 m/z). Dynamic

18

exclusion (30 s) was on.

The

MS

analysis

was

performed

on

a

Q-Exactive

Hybrid

19

The raw data files generated by the Q-Exactive were analyzed with software

20

MaxQuant version 1.3.0.5, which was developed by Matthias Mann’s lab.19 The

21

MS/MS spectra were searched against the Human Uniprot FASTA database. Trypsin

22

specificity (KR/P, DE) was adopted, and up to two missed cleavage sites were


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1

allowed. Carbamidomethyl (C) was chosen for fixed modifications, and oxidation on

2

methionine, phospho (S, T, Y) were set as variable modifications. The mass tolerances

3

for the precursor ions and fragment ions were set to 10 ppm and 0.05 Da, respectively.

4

The maximum false-discovery rate (FDR) was set to 1 % for both the peptides and

5

proteins. All the phosphorylation sites reported in this study met the combined cutoff

6

values of localization probability >0.75 and ΔPTM score ≥5. Sequence logos were

7

automatically generated by the WebLogo (http://weblogo.berkeley.edu/logo.cgi).20

8

RESULTS AND DISCUSSION

9

In this study we aimed to fractionate the pTyr peptides according to their binding

10

affinity to Src SH2 superbinder. This was achieved by eluting the bound pTyr peptides

11

with increased concentrations of a competing agent. The peptide pYEEI was found to

12

have the strongest affinity to the Src SH2 domain by screening a degenerate

13

phosphopeptide library.14-16 Because the SH2 superbinder and the wild-type SH2

14

domain have similar specificity toward pTyr peptides, pYEEI would be a good

15

competing reagent for selective elution of bound pTyr peptides. However, the

16

presence of pYEEI in the eluted sample would interfere with subsequent LC-MS/MS

17

analysis of pTyr peptides. To solve this problem, we used biotin-pYEEI as the

18

competing agent. After elution, the biotin-pYEEI could be removed by immobilized

19

NeutrAvidin. Thus, in this approach two affinity purifications are performed. To

20

facilitate

21

chromatography scheme as shown in Figure 1. The sample to be enriched was first

22

incubated with the Sepharose beads with immobilized Src superbinder at 4°C

this fractionation

procedure,

we

designed

the

biphasic

affinity



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Page 10 of 23

1

overnight with rotation. After the pTyr peptides were bound to the superbinder, the

2

beads were transferred into a 1mL-pippet tip prepacked with NeutrAvidin agarose

3

resins at the end of the tip. In this way, a biphasic affinity tip, where the superbinder

4

was at the top and the avidin was at the bottom, was formed. When the biotin-pYEEI

5

was used to elute the bound pTyr peptides from the superbinder beads, the excess

6

biotin-pYEEI was directily captured by the avidin at the bottom of the tip. In this way,

7

the enriched pTyr peptides would be free of biotin-pYEEI. Using the biphasic affinity

8

tip, the fractionation of the pTyr peptides could be conveniently achieved by elution

9

with increasing concentrations of competitive agent biotin-pYEEI.

10

We first investigated the performance of this approach to enrich pTyr peptides

11

from the complex peptide mixture. Tryptic digest of the total cell lysate of Jurkat cells

12

was first subjected to purification with immobilized titanium (IV) ion affinity

13

chromatography (Ti4+-IMAC) to deplete unphosphorylated peptides. The resulting

14

phosphopeptides, with the majority of them being pSer/pThr peptides as they were the

15

highly abundant ones, were then subjected to the biphasic affinity purification scheme

16

to enrich the low-abundance pTyr peptides. The biphasic affinity tip was thoroughly

17

washed to remove pSer/pThr peptides and residual nonphosphorylated peptides. The

18

bound pTyr peptides were then eluted by 30 µM biotin-pYEEI and analyzed by

19

LC-MS/MS. This resulted in the identification of 2,467 unique peptides

20

(Supplementary Table 1), of which 2,194 (88.93%) were pTyr peptides, indicating

21

good specificity. We also investigated if the avidin purification could be omitted. As

22

shown in Figure 2A, the biotin-pYEEI was observed to be eluted from 65 min to 90


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1

min for the RPLC analysis of the sample without avidin purification. Because of

2

serious interference of the biotin-pYEEI in the sample, only 1,300 pTyr peptides were

3

identified without NeutrAvidin. As shown in Figure 2B, the biphasic affinity

4

chromatography could effectively remove the interference of biotin-pYEEI, resulting

5

in a 69% increase in pTyr peptide identification. Clearly the biphasic affinity tip with

6

NeutrAvidin was preferred for competitive elution.

7

Instead of competitive elution, the bound peptides could also be eluted by TFA,

8

which resulted in the identification of 3,034 unique peptides where 2,553 (84.15%)

9

were pTyr peptides. Compared with TFA elution, higher specificity was obtained by

10

the competitive elution. However, a slightly lower number of pTyr peptides was

11

identified by the latter approach. Similar results were obtained from replicate runs

12

(Supplementary Table 1). Combining the pTyr sites identified from two replicate runs

13

for each method, the competition method recapitulated 82% of the pTyr sites

14

identified by the TFA elution method. The number of identified pTyr sites did not

15

increase with a further increase of the biotin-pYEEI concentration, indicating that 30

16

µM competitive peptide was sufficient to elute the bound pTyr peptides from the

17

superbinder (Supplementary Table 1). A significant advantage of the competitive

18

approach is its high specificity. When the crude protein digest without Ti4+-IMAC

19

enrichment was directly subjected to the biphasic affinity chromatography

20

purification, high specificity (>72%) was still achieved (Supplementary Table 1). In

21

contrast, for the TFA elution method, the specificity for pTyr identification was 44%

22

with the majority of identified peptides found to be unphosphorylated.



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1

To achieve wider coverage of the Tyr phosphoproteome, we used step-wise

2

competitive elution to fractionate the bound pTyr peptides in the biphasic affnity

3

purification protocol. The same sample, i.e., the phosphopeptides enriched from the

4

digest of the total cell lysate of Jurkat cells by Ti4+-IMAC, was loaded onto the

5

superbinder sepharose beads. The weakly bound peptides were eluted by washing

6

with low concentration (4 µM) of biotin-pYEEI. More strongly bound peptides were

7

eluted stepwise with 8 µM, 16 µM and 32 µM biotin-pYEEI. Each fraction was

8

seperately collected and analyzed by LC-MS/MS. After database searching, 3,480

9

unique pTyr sites were identified in 2 replicate MS runs from 2 mg initial Jurkat cell

10

lysate. We compared the number of identified unique pTyr sites in different fractions

11

(Figure 3A). It was found that most pTyr sites, 1,467 (42.2 %), were identified in

12

fraction 2. Only 62 (1.8 %) were detected in all the four fractions indicating excellent

13

resolution of fractionation. For deep analysis of tyrosine phosphoproteome, separation

14

and fractionation approaches to further reduce the sample complexity are equally

15

important with specific enrichment. As shown in Figure 3B, compared with the single

16

step competitive elution method, 2.2 folds more pTyr sites were identified by the

17

fractionation method, which indicated the coverage was improved significantly. We

18

also found that >85% of sites identified by the TFA elution could also be identified by

19

the fractionation method, indicating that the step-wise elution with the competitive

20

peptide was fairly complete.

21

According to the mechanism of competitive elution, the weakly bound peptides

22

would be firstly eluted with the initial elution buffer containing low concentration of


Page 13 of 23

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1

biotin-pYEEI, and more strongly bound peptides would be eluted in the next round of

2

wash with higher concentration of elution agent. As the binding of Src superbinder to

3

pTyr peptides is dependent on the primary sequence around the pTyr, we compared

4

the amino acid residues around the pTyr sites identified in the four sequential

5

competitive elutions to elucidate the characteristic distribution. First, we investigated

6

the consensus sequences containing pTyr sites using WebLogo software for the four

7

fractions.20 Considering the weakly bound peptides may not be completely replaced

8

by the elution agent and still appeared in the latter eluent, we separately uploaded the

9

new identified pTyr sites from each elution compared to the results in the former

10

elutions with 13-amino acid sequence window to WebLogo and obtained the

11

frequency plots as shown in Figure 4. An apparent increase in frequencies for Asp (D)

12

and Glu (E), C-terminal to the pY residue (pY+1, +2), were observed in the four

13

sequential elutions, indicating these two residues are dominant determinants of strong

14

binding affinity to the Src superbinder. Besides, sequences containing Leu (L)/Val

15

(V)/Pro (P)/Ile (I) occupied a great proportion at position of pY+3 for the strong

16

binding. Consequently, the Src superbinder preferred phosphopeptides with

17

pY-[D/E]-[D/E]-[L/I/V/P] motif. This motif is highly consistent with the binding

18

motif of wide type Src SH2 domain, which confirms that superbinder SH2 has the

19

same specificity regardless of its markedly enhanced binding affinity.

20

The pTyr mediates the assembly of protein complexes essential to intracellular

21

kinase signaling. Comprehensive Resource of Mammalian Protein Complexes

22

(CORUM) is a database of manually curated and validated mammalian protein



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Page 14 of 23

1

complexes.21 The pTyr sites identified in this study were mapped to the complexes in

2

CORUM. Among 1,565 complexes in the CORUM, 538 (34.4 %) contain at least 1

3

pTyr sites (Supplementary Figure 1). Some complexes (98, 6.3 %) have more than 5

4

pTyr sites. This indicated that tyrosine kinases play a significant role in regulating the

5

function or assembling of protein complex. The fractionation of pTyr peptides by the

6

Src SH2 superbinder enabled the separation of the pTyr sites into different categories.

7

We then investigated if any macromolecular complexes enriched with pTyr proteins

8

with different site categories. It was found a total of 86 complexes were enriched with

9

p-value

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