SH2 Superbinder Modified Monolithic Capillary Column for the

Subscriber access provided by READING UNIV

Article

SH2 superbinder modified monolithic capillary column for the sensitive analysis of protein tyrosine phosphorylation Yating Yao, Yangyang Bian, Mingming Dong, Yan Wang, Jiawen Lv, Lianfang Chen, Hongwei Wang, Jiawei Mao, Jing Dong, and Mingliang Ye J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00546 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

SH2 superbinder modified monolithic capillary column for the sensitive analysis of protein tyrosine phosphorylation

Yating Yao1,2,4, Yangyang Bian1,3,4, Mingming Dong1,5,*, Yan Wang1,2, Jiawen Lv1,2, Lianfang Chen1,2, Hongwei Wang1,2, Jiawei Mao1,2, Jing Dong1, Mingliang Ye1,*

1

CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National

Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; 2

University of Chinese Academy of Sciences, Beijing 100049, China;

3

Medical Research Center, The First Affiliated Hospital of Zhengzhou University,

Zhengzhou University, Zhengzhou, Henan 450052, China; 5

Dalian Ocean University, Dalian 116023, China;

4

These authors contributed equally to the work.

*

To whom correspondence should be addressed:

*Phone: +86-411-84379620. Fax: +86-411-84379620. E-mail: [email protected]. (M.M. Dong) *Phone: +86-411-84379610. Fax: +86-411-84379620. E-mail: [email protected] (M.L.Ye)

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In this study, we presents a method to specifically capture phosphotyrosine (pTyr) peptides from minute amount of sample for the sensitive analysis of protein tyrosine phosphorylation. We immobilized SH2 superbinder on a monolithic capillary column to construct a microreactor to enrich pTyr peptides. It was found the synthetic pTyr peptide could be specifically enriched by the microreactor from the peptide mixture prepared by spiking of the synthetic pTyr peptide into the tryptic digests of α-casein and β-casein with molar ratios of 1:1000:1000. The microreactor was further applied to enrich pTyr peptides from pervanadate-treated HeLa cell digests for phosphoproteomics analysis, which resulted in the identification of 796 unique pTyr sites. In contrast, the conventional SH2 superbinder-based method identified 41 pTyr sites for the same sample, only 5.2% of the number achieved by the microreactor. Finally, this microreactor was also applied to analyze the pTyr in Shc1 complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Bring together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only limited amount of sample is available. Key words：： Phosphoproteomics, SH2 superbinder, monolithic capillary column, protein tyrosine phosphorylation, peptides, phosphopeptides, enrichment, microreactor, LC−MS/MS, protein complex

2


Page 2 of 28

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Protein tyrosine phosphorylation (pTyr) is a crucial cell signaling mechanism that regulates many important biological processes such as proliferation, differentiation, hormone

responses,

as

well

as

immune

defense(1-4).

Aberrant

tyrosine

phosphorylation often results in some prevalent cancers(5, 6). So the ability to identify the unregulated tyrosine phosphorylations that drive tumor survival and growth plays a crucial role in the development of cancer diagnostics and therapeutics. However, due to the relatively low cellular levels of tyrosine phosphorylation compared to serine and threonine phosphorylation (pSer/pThr) events, most phosphoproteomic approaches reveal only small numbers of tyrosine phosphorylation sites. To address this underrepresentation issue, many techniques and strategies have been developed to specifically isolate and enrich tyrosine phosphopeptides from a mixture before mass spectrometry (MS) analysis. A typical technique for pTyr peptide enrichment makes use of commercially available anti-pTyr antibodies conjugated to beads(7, 8). For instance, Abe et al. used antibody of pY1000 to enrich pTyr peptides which leaded to identification of more than 1000 pTyr sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment (9). Although the antibodies show high specificity for pTyr recognition, the high cost often hinders their use in quantities sufficient to saturate the pTyr peptides in a sample, resulting in a poor reproducibility and a relatively low coverage of the Tyr phosphoproteome(10). Approaches based on molecular imprinted polymers (MIPs) were also developed for the enrichment of pTyr peptides(11, 12). However, the complexity of the synthetic process, low adsorption and low selectivity make the routine application of these methods in Tyr phosphoproteomics a challenge. Consequently, it is essential to establish a more efficient and cost-effective approach for the deeper identification of Tyr phosphoproteome. Src homology 2 (SH2) domain, a sequence-specific phosphotyrosine binding module dedicated to the recognition of phosphorylated tyrosine residues in proteins, is a potential affinity reagent for pTyr peptides(13-15). However, the application of wild 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

type SH2 domains in Tyr phosphoproteomics is severely limited by their modest affinities. SH2 variants containing three amino acid substitutions, i.e., the SH2 superbinder, were then designed to have markedly enhanced affinities for pTyr peptides than nature SH2 domain(16, 17). By exploiting this high affinity, we developed a method in which the SH2 domain-derived pTyr-superbinder was chelated on Ni2+-NTA beads via its His tag as the affinity reagent for the pTyr peptide enrichment(18). It is demonstrated that the superbinder is much more economical, and surpasses the performance with anti-pTyr antibodies. Indeed, about 20,000 unique pTyr peptides and more than 10,000 pTyr sites were identified from nine human cell lines, achieving unprecedented deep coverage for tyrosine phosphoproteome analysis. However, two desalting processes were required in this method, which made this method tedious and cumbersome. Recently, a seamless workflow using Ti4+-IMAC and covalently immobilized SH2 superbinder was developed. Compared with the previous method, this method can identify 41% more pTyr peptides(19). We also developed a biphasic affinity chromatographic strategy for tyrosine phosphoproteome analysis, where

SH2

superbinder was coupled with NeutrAvidin affinity

chromatography and biotin-pYEEI was used as the competitive reagent to elute pTyr peptides from the SH2 superbinder. According to their binding affinities to SH2 superbinder, the pTyr peptides could be fractionated when stepwise elution with biotin-pYEEI of different concentrations was applied(20). However, these methods are not fitted to analyze minute amount of sample. A monolithic column is an interesting option for the preparation of microreactor to process minute amount of sample for proteomics analysis as it is easy to fabricate and the porous structure provides high surface area and fast mass transfer rate(21, 22). For instance, enzyme-immobilized monolithic microreactor was prepared to efficiently digest minute amount of proteins or genomic DNA(23-25), immobilized metal ion affinity chromatography with silica or polymer monolithic capillary column was developed to enrich phosphopeptides(26), and peptide-N-glycosidase F was immobilized onto a porous polymer-based monolith to remove N-glycans from small 4


Page 4 of 28

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and medium size glycoproteins(27). These studies have demonstrated the monolithic microreators were beneficial to improve the detection sensitivity. In this study, we immobilized SH2 superbinder onto a monolithic capillary column to construct a microreactor to enrich pTyr peptides from the minute amount of sample. This method takes full advantage of the small scale of the monolithic capillary microreactor and combines it with SH2 superbinder which provides high affinity towards phosphorylated tyrosine residues for subsequent tyrosine phosphoproteome analysis. This combination solves the problems of lower sensitivity in the method where SH2 modified sepharose beads are used. By using this technique, we identified more than 700 pTyr sites from only 100 µg phosphopeptide sample purified from pervanadate-treated HeLa cell digests. This approach was also successfully applied to the analysis of the pTyr in a protein complex, which further demonstrated the applicability of our method for the analysis of minute amount of sample.

Experiment section Materials and chemicals Fused-silica capillaries with 200 µm or 75 µm inner diameter were obtained from PolymicroTechnologies

(Phoenix,

AZ,

USA). Ethyleneglycol

dimethacrylate

(EDMA), glycidyl methacrylate (GMA), azobisisobutyronitrile (AIBN), cyclohexanol, 1-dodecanol, sodium cyanoborohydride, and glycine were obtained from Aldrich (Milwaukee, WI, USA). Ammonia solution (NH3H2O, 25%) and acetonitrile were provided by Merck (Darmstadt, Germany). Trifluoroacetic acid (TFA), dithiothreitol (DTT), iodoracetamide (IAA), sodium chloride (NaCl), glutaraldehydesolution (50%), sodium azide, γ-methacryloxypropyltrimethoxysilane (γ-MAPS), α-casein, β-casein and trypsin (bovine, TPCK-treated) were obtained from Sigma(St. Louis, MO, USA). Formic acid (FA) was purchased from Fluka (Buches, Germany). Other chemicals and reagents were either of analytical grade or of better grade. High-purity water used in all of the experiments was obtained from a Milli-Q purification system (Millipore, Bedford, MA). Preparation of Monolithic Capillary Columns 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The vinylization of the inner wall of the capillary was performed as reported(28, 29). The capillary was first rinsed with a 1 M NaOH solution for 1 h, and then rinsed with water and methanol for 10 min, respectively. After drying the capillary with nitrogen gas, the capillary was filled with γ-MAPS methanol solution (50%, v/v) and kept at room temperature for one day. Finally, the capillary was flushed with methanol and dried with nitrogen gas. The polymerization mixture was composed of 0.3 mL GMA, 0.1 mL EDMA, 1.1 mL cyclohexanol, 0.1 mL 1-dodecanol, and 4.0 mg AIBN(30). The solution was degassed with nitrogen and sonicated for 15 min. Subsequently, the solution was manually filled into the pretreated capillaries (30 cm length) by a syringe. Both ends of the capillaries were sealed with silicon rubbers, and the polymerization was processed at 50 °C in a water bath for 12 h. After the polymerization was complete, each column was inspected under a light microscope to check the homogeneity of the monolith. Methanol was pumped through the monoliths to wash out the porogenic solvents and other compounds. As shown in Figure S1, the SH2 superbinder was immobilized on the poly (glycidylmethacrylate-co-ethylene dimethacrylate) monolithic support, according to an existing procedure with slight modifications(30). The monolithic columns were first flushed with water for 30 min, followed by 25% (v/v) ammonium hydroxide solution. Both ends of the columns were sealed with silicon rubbers, and the amination reaction was processed at 40 °C in a water bath for 3 h. After a 2 h wash with a 0.1 M borate buffer (pH 8.2), a 2.5% (v/v) glutaraldehyde solution in the borate buffer was pumped through the columns at room temperature for 2 h. The columns were then equilibrated by pumping 1 x PBS (pH 7.4) for 2 h. After that, the SH2 superbinder was coupled to the support by continuously introducing 2.0 mg/mL SH2 superbinder dissolved in 1 x PBS (pH 7.4) at 4 oC overnight. Finally, the SH2 superbinder immobilized monolithic columns were flushed with 0.1M Tris-HCl, 0.75 M glycine solution (pH 8.3) at room temperature for 2 h. To prevent leaching of SH2 6


Page 6 of 28

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


superbinder from the column, the labile Schiff base was reduced with an 80 mM sodium borohydride solution (6 h) to form a stable secondary amine (see Figure S1). Finally, the capillaries were filled with immunoaffinity purification (IAP) buffer containing 50 mM 3-morpholinopropanesulfonic acid (MOPS) (pH 7.2), 50 mM NaCl, 10 mM Na2HPO4 and 0.05% NaN3. Then the capillaries were cut to a length of 10 cm and stored at 4 °C before use. All columns used in this work were disposable. Further investigation of the monolithic support was performed via scanning electron microscopy (SEM), using a JEOLModel JSM-6701F scanning electron microscope (JEOL, Tokyo,Japan). Cell culture and digest preparation The HeLa cells were grown in RPMI 1640 medium, supplemented with 10% new born bovine serum, 100 µg/mL of streptomycin and 100 U/mL of penicillin in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were harvested until 80% confluence, and then treated with 2.5 mM freshly prepared pervanadate for 15 min at 37 °C(18, 31). After that, the cell pellets were gently homogenized in an ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 8 M urea, phosphatase inhibitor (1 mM NaF, 1 mM Na3VO4, 1 mM C3H7Na2O6P, and 10 mM Na4O7P2), 1% Triton X-100 (v/v) and 2% protease inhibitor cocktail (v/v) in ice bath, sonicated at 400 W for 10 min, and centrifuged at 25, 000 g for 30 min. The supernatant was precipitated in ice-cold acetone/ethanol/acetic acid (50/50/0.1, v/v/v). Then the obtained proteins were dissolved in reducing buffer containing 8 M urea and 50 mM Tris-HCl (pH 8.0), and the final protein concentration was determined by Bradford assay. The redissolved proteins were reduced by 20 mM DTT and alkylated by 40 mM IAA, then diluted to 1 M Urea by 50 mM Tris-HCl buffer (pH 8.2). Trypsin digestion was performed by incubation protein and trypsin overnight at 37 °C with a ratio of enzyme-to-protein at 1:20 (w/w). The obtained peptide mixture was kept at −80 °C for further usage. α-casein and β-casein were digested as the aforementioned procedures(32). Phosphotyrosyl peptide enrichment by SH2 superbinder modified monolithic 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

column For the enrichment of phosphotyrosyl peptide from a semicomplex sample originating from tryptic digests of α-casein, β-casein and commercial standard phosphotyrosyl peptide, the procedures were as follows. Before loading samples, the SH2 superbinder modified monolithic columns were equilibrated with IAP buffer, then the peptide mixture dissolved in IAP buffer were manually filled into the columns to achieve complete binding. Afterwards, the columns were washed with IAP buffer to remove the non-specific binding peptides. Eventually, the bound peptides were eluted by 0.2% TFA, dried down using a Speed-Vac and saved at -20 oC until further usage. A pre-enrichment procedure of phosphopeptides is needed before performing the enrichment of phosphotyrosyl peptides from HeLa cell digests. And the enrichment of phosphopeptides was performed according to that described before with some modifications(32). Briefly, 1 mg Hela cell digests (1 mL) were mixed with identical volume of loading buffer (80% ACN/6% TFA), and then the mixture were loaded on SPE column packed with 20 mg Ti4+-IMAC adsorbents. After that, the column was washed with 50% ACN/200 mM NaCl/6% TFA and 30% ACN/0.1%TFA, respectively. Finally, the bound peptides were eluted using 10% NH3H2O and dried down using a Speed-Vac. Different amount of the enriched phosphopeptides, e.g. 1/10 of the eluted sample equivalent to 100 µg starting materials, were subjected to pTyr peptide enrichment using SH2 microreactor with the same procedures as introduced above. In the control experiments, the pTyr peptide enrichment using SH2 sepharose beads proceeded as the protocols reported by Dong et al(19). MALDI TOF/TOF analysis All the MALDI-TOF mass experiments were performed using AB Sciex 5800 MALDI-TOF/TOF mass spectrometer (AB Sciex, CA) equipped with a pulsed Nd/YAG laser at 355 nm. All spectra reported were obtained in the reflex positive ion mode with delayed ion extraction. DHB (25 mg/mL) in 50% acetonitrile, 1% phosphoric acid was used as the matrix. Aliquots of the sample (0.5 µL) and DHB 8


Page 8 of 28

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


matrix solution (0.5 µL) were sequentially spotted on the MALDI plate and dried at room temperature prior to MALDI-TOF MS analysis. Liquid chromatography-tandem mass spectrometry analysis The lyophilized peptide samples were dissolved in 0.1% FA/H2O solution and manually loaded onto a 15 cm capillary analysis column (75 µm i.d.) for mass spectrometry analysis. The LC−MS/MS system consisted of an Agilent 1100 HPLC system and an LTQ–Orbitrap Velos mass spectrometer. The peptides were separated by using a gradient of 130 min, where 0.1% FA in water and 0.1% FA in acetonitrile were used as the mobile phase A and B, respectively. The flow rate was ~ 300 nL/min after splitting. The mass spectrometry was operated in positive ion mode with a capillary temperature of 250 °C. The full mass scan spectra (m/z 400 – 2000) were acquired in the Orbitrap mass analyzer with a resolution of 60 000. The 15 most intense ions from the full scan were fragmented by collision-induced dissociation (CID) in the LTQ. The dynamic exclusion function was set as follows: repeat, 2; duration, 30 s; exclusion duration, 60 s. System control and data collection were carried out by Xcalibur software. In the Shc1 experiment, the LC-MS/MS analysis was performed using a Dionex UltiMate 3000 RSLCnano system (Thermo Scientific, USA) with a Q-Exactive mass spectrometer (Thermo Scientific, USA). The sample was loaded onto the a 3 cm C18 trap column (200 µm i.d.) at a flow rate of 5 µL/min with 0.1% formic acid (FA) as loading buffier. The analytical column (75 µm i.d.) was packed with C18 particles (3 µm) to 15 cm length. 0.1% FA in water and 0.1% FA in 80% acetonitrile were used as the mobile phase A and B, respectively, and the elution gradient executed was 5% to 35% mobile phase B lasted for 78 min. The Q Exactive mass spectrometer was operated in data-dependent mode. Full mass scan MS spectra (m/z 400 −2 000) were acquired by the Orbitrap mass analyzer with a resolution of 70 000 (m/z 200). The 12 most intense multiply charged ions (charge states ≥ 2) were fragmented by higher-energy collisional dissociation (HCD). The MS/MS scans were also acquired by the Orbitrap mass analyzer with a 35 000 resolution (m/z 200), and the AGC target 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was set to 1×105 with a max injection time of 120 ms. The temperature of the ion transfer capillary was set as 275 oC. Other mass spectrometric settings were as follows: spray voltage, 2.0 kV; normalized HCD collision energy, 27%; dynamic exclusion, 30 s. Database searching and peptide identification. The MS raw data files were searched by MaxQuant(33) against a nonredundant Uniprot human database (containing 70 946 sequences, and downloaded from website of www.uniprot.org). Carbamidomethyl cysteine was set as a fixed modification, phosphorylation of serine, threonine, and tyrosine and oxidation of methionine were allowed as variable modifications. Trypsin was specified as the proteolytic enzyme with up to three missed cleavage sites allowed. Parent ions mass tolerance was set as 10 ppm, and fragment ions mass tolerance was 0.05 Da. The false discovery rate (FDR) was controlled to < 0.01 at both peptide and protein level. The other settings were the same as the conventional search. The obtained phosphorylated sites were further filtered by Localization Probability ≥0.75 and Score Difference ≥5, which means high confident (Class I) of phosphorylated sites. Sequence logos were automatically generated by the WebLogo (http://weblogo.berkeley.edu/logo. cgi)(34). All the mass spectrometry data have been deposited to the jPOST database (http://jpostdb.org/). Accession code: JPST000336/PXD007998.

Results and Discussion SH2 superbinder modified sepharose beads are great for the analysis of tyrosine phosphoproteome when sufficient sample is available(19). However, this method is not applicable to process minute amount of sample. The capillary column format can facilitate the processing of minute amount of sample. For this reason, we also attempted to pack the sepharose beads into a capillary with the i.d. of 200 µm, but failed due to the big size and poor mechanical stability of sepharose beads. It is well known that the monolithic capillary columns have the unique features to process minute amount of sample. Especially, the diffusion in monolith is enhanced by 10


Page 10 of 28

Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


convection due to the presence of through pores while in the beads packed column the analytes must diffuse into the small pores from the main stream of the mobile phase as it flows around the packing particles(35). For this reason, we decided to prepare monolithic affinity chromatography capillary column. As the porous polymer monoliths polymerized from GMA and EDMA has the advantages of high hydrophilicity and easy modification(30, 36), we immobilized SH2 superbinders onto this monolith to enrich pTyr peptides for phosphoproteome analysis. As shown in Figure S1, the monolith was in situ synthesized in a fused-silica capillary (i.d. 200 µm) using GMA as the monomers, EDMA as the crosslinker, cyclohexanol and dodecyl alcohol as the porogen and AIBN as the initiator. Then ammonium hydroxide was used to introduce amino groups to the surface of the capillary monolith. Finally, the SH2 superbinder was immobilized onto the monolith via Schiff-Base reaction by using glutaraldehyde as the linker. To enhance the stability of immobilized SH2 superbinder, sodium cyanoborohydride was used to reduce the formed C=N to stable C−N bonds. The constructed monolith was characterized by scanning electron microscopy (SEM). As shown in Figure 1, the capillary column is fully filled with the homogenous monolithic matrix, and the matrix with porous structure was tightly anchored to the inner wall of the capillary without any disconnection. Due to the existence of large through-pores, the monolithic capillary column has good permeability and low back pressure, enabling peptide solution be directly injected into capillary monolithic column by just hand pushing or a low-pressure microinjection pump. Additionally, the macropore structure would provide large surface area of the monolithic matrix, which enables the high density binding of superbinder. The total volume of the SH2 superbinder modified monolithic capillary column used in this work was about 3 µL, which facilitate the enrichment of pTyr peptides from minute amount of sample. As the SH2 superbinder modified monolithic capillary microreactor, herein after to 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

be referred as SH2 microreactor, is a combination of monolithic capillary column and SH2 superbinder, we speculate that the SH2 microreactor can integrate the advantages of high sensitivity from monolithic capillary column and high affinity from SH2 superbinder. And to verify our speculation, we applied SH2 microreactor to capture the synthetic pTyr peptide that we spiked into the tryptic digests of α-casein and β-casein with different molar ratios. After washing with IAP buffer, the bounded pTyr peptide was eluted by 0.2% TFA and subsequently submitted to MALDI-TOF MS analysis. As a control, the peptide mixture without SH2 microreactor enrichment was also analyzed by MALDI. As shown in Figure 2a, for the direct analysis, many peaks were observed in the mass spectrum, where the pTyr peptide cannot be clearly recognized. After enrichment, the pTyr peptide dominated the mass spectrum, and no obvious non-specific binding peptides were observed (Figure 2b). Even the molar ratio increased to 1:500:500 or 1:1000:1000, the peak of pTyr peptide was still obvious with fewer non-specific binding peptides (Figure 2c-d). This result clearly indicated that the pTyr peptide can be selectively enriched from the peptide mixture using our prepared SH2 microreactor. The major objective of this study is to develop a new sensitive platform for pTyr peptide enrichment. Then the sensitivity of the platform by SH2 microreactor enrichment was investigated by analyzing the peptide mixtures comprised of the tryptic digests of α-casein and β-casein with different amounts of synthetic pTyr peptide (the molar ratios of pTyr peptide : α-casein : β-casein were kept at 1 : 1 : 1 ). As shown in Figure 3, the pY peptide were successfully enriched by the SH2 microreactor in 5, 1 and 0.1 fmol peptide mixture. These results demonstrated that the SH2 microreactor has the potential to process minute amount of sample. After demonstrating the feasibility of the SH2 microreactor to isolate pTyr peptide from peptide mixture, a more complex sample, HeLa cell digest, was used to test the performance of the SH2 microreactor. Due to the ultra-low abundance of pTyr peptides and high complexity of the sample, the cell digest was firstly subjected to 12


Page 12 of 28

Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ti4+-IMAC enrichment for phosphopeptides and then subjected to SH2 enrichment for pTyr peptides (if not otherwise stated, the flowing descriptions “starting material” are referred to the phosphopeptide sample purified from pervanadate-treated HeLa cell digests). In our previous work where SH2 superbinder modified sepharose beads were used, the minimum starting material was 2 mg phosphopeptide samples(19). Then in this work targeting minute sample analysis, the starting amount we used for SH2 microreactor enrichment is no more than 1 mg. Both the SH2 modified sepharose beads and the SH2 microreactor were used to analyze the tyrosine phosphoproteome of 1 mg HeLa cell digest. It was found only 773 pTyr sites (if not otherwise stated, the sites were combined by two replicate runs) were identified when SH2 modified sepharose beads were used, while this number increased to 1084 (increased by 40%) in SH2 microreactor enrichment method (Table S1). This result suggested that the SH2 microreactor may be a better option for the enrichment of pTyr peptides from small amount of samples. To validate this assumption, we further decreased the amount of starting materials to 500 µg, 200 µg and 100 µg to compare the enrichment performance of SH2 sepharose beads and SH2 microreactor. The identifications were shown in Figure 4 and Table S1, more pTyr sites were obtained from SH2 microreactor strategy, and with the decrease of starting sample amount, the superiority of SH2 microreactor became even more pronounced. Still 796 pTyr sites were identified from 100 µg samples in SH2 microreactor method, however, the corresponding number of pTyr sites is only 41 in SH2 sepharose beads method. Clearly, SH2 microreactor was preferred for the enrichment of pTyr peptides from minute amount of sample. Moreover, it is essential to emphasize that the enrichment specificity is relatively high in both methods with starting materials range from 100 to 1000 µg, which fully indicates the high affinity of SH2 superbinder towards pTyr peptides. We then investigated the distribution of amino acid residues around the pTyr sites identified by the two methods. The seven amino acids up- and down-stream of the pTyr sites were submitted for the WebLogo analysis(34). As presented in Figure S2, the amino acid sequences surrounding pTyr sites were similar between these two dataset, with D, E overpresented on +1 position. This is not surprising since the same 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

affinity reagent was used for the both methods. The identification overlaps between the two methods were also examined. We take the identifications obtained from 100 µg and 1 000 µg sample as examples. As shown in Figure S3, a large proportion (85% and 69%, corresponding to 100 µg and 1 000 µg sample, respectively) of pTyr sites identified in SH2 sepharose beads method was also identified in SH2 microreactor method, further indicating the higher recovery of SH2 microreactor method for small amount of sample analysis. We then searched the tyrosine phosphorylated proteins identified from 100 µg starting materials using SH2 microreactor and SH2 sepharose beads against PaxDb database(37), a protein abundance database, to investigate their abundances in HeLa cell lines. The data (Figure S4) illustrated that most of the identified pTyr sites were from the high abundance proteins in both methods, and only a small proportion of proteins ranked at or under the bottom 25%. However, about 2.8% proteins ranked at bottom 10% were still identified in SH2 microcreator method, indicating its higher sensitivity than that of SH2 sepharose beads method when processing small amount of sample. The reproducibility of the SH2 microreactor was then investigated. Four different SH2 microreactors were prepared to process the same amount of sample (100 µg phosphopeptides purified from pervanadate-treated HeLa cell digests) in parallel. The enriched pTyr peptides were eluted from the SH2 microreactors and submitted to LC-MS/MS analysis. The pTyr site identifications were summarized in Figure S5. Totally, 823 pTyr sites were identified by combining the identification results, and 482 (RSD=15.99%, n=4) pTyr sites were averagely identified with an enrichment specificity of 87.43% (RSD=2.96%, n=4). The results demonstrated that the reproducibility of SH2 microreactor was a little bit poor than that of SH2 sepharose beads, however, it was much more excellent than that of pY99 antibodies used in a pTyr immuno-affinity purification (pY-IP) experiment (RSD=42%, n=6)(19, 38). We finally applied our SH2 microreactor to capture pTyr peptides from a protein complex containing Shc1 and its interacting proteins. Shc1, also termed as ShcA, 14


Page 14 of 28

Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


plays a physiological role in coupling activated receptor tyrosine kinases (RTKs) to promote downstream signaling events(39-41). And it has been demonstrated that Shc1 responds to epidermal growth factor (EGF) stimulation through multiple distinct phosphorylation events and protein-protein interactions(42). However, it is very challenging to analyze the phosphorylation of the protein complex, particularly the tyrosine phosphorylation. One of the major reasons is that the amount of a protein complex obtained from an immunoprecipitation experiment is very small, usually only a dozen microgrammes. And the ultra-low abundance of tyrosine phosphorylation than that of serine/threonine phosphorylation makes it even harder to isolate pTyr peptides from the peptide mixture generated from the protein complex. By combining affinity capture using antibodies to Shc1 with mass spectrometry, Dyson et al. identified seven known binding partners and two known Shc1 phosphorylation sites (only one is pTyr site, SHC1 Y313) in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells(43). In this study, we first co-immunoprecipitated (co-IP) Shc1 protein complex with anti-Flag antibodies from the total cell lysate of HeLa cells after 2 min EGF stimulation. The enriched protein complex was digested by trypsin. After redissolved in IAP buffer, the digest was submitted to SH2 microreactor for the enrichment of pTyr peptides. After the washing and elution procedures, the obtained pTyr peptides were analyzed by LC-MS/MS. The proportion of pTyr sites in all phosphorylation sites increased from 22.1% (15/68) before enrichment to 77.8% (21/27) after enrichment, which indicated the high specificity of our SH2 microreactor toward tyrosine phosphorylated peptides. The identifications of pTyr sites belonging to Shc1 complex were detailed in Figure 5. The number of identified pTyr site increased from 9 before enrichment to 15 after enrichment (Figure 5a). As shown in Figure 5b, three pTyr sites presented in the EGFR (Y1197, Y1110, Y1172) were successfully identified before enrichment, however, with the enrichment by SH2 microreactor, another EGFR pTyr site, Y1092 could as well be identified. According to Zheng’s work(42), the phosphorylation of Y1197, Y1110, Y1172 in EGFR reached a maximum at 2 min after EGF addition, while there was no information about the pY1092. We supposed that the relative 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

abundance of pY1092 might be lower than that of the other three identified pTyr sites in EGFR, then it was not so easy to be identified without any enrichment. According to the PhosphositePlus dataset(44), we investigated the number of records in which all of the identified pTyr sites were assigned using only proteomic discovery-mode mass spectrometry (Figure 5c). It was found the record number of pY1197, pY1110 and pY1172 was 1040, 457 and 991, respectively, while the corresponding record number of pY1092 was only 358. Generally speaking, a phosphorylation site with relative high abundance or high ionization efficiency is more easily to be identified in the proteomic discovery-mode mass spectrometry. Thus the lower record number indicated lower abundance or low ionization efficiency of the pTyr peptides. Clearly the enrichment might improve the sensitivity to identify pY1092 site on EGFR in Shc1 complex. The similar situations also occurred on pY74 and pY453 on GAREM1, and pY659 and pY627 on GAB1, where GAREM1 pY74 and GAB1 pY627 site could be only identified after enrichment while before enrichment could not. Besides EGFR pY1092, GAREM1 pY74, the other five pTyr sites were also detected in the Shc1 complex sample after SH2 microreactor enrichment (Figure 5b). The record numbers of pTyr sites shown in Figure 5c may not correlate well with their abundance in the Shc1 complex. However, all of the 16 sites were observed at least dozens of times (the lowest record number is 42 for GAREM1 pY74), indicating high confidence of these identifications. To our knowledge, this is the highest number of pTyr sites identified from Shc1 complex. Clearly, the SH2 microreactor is fitted to enrich pTyr peptides from an IP sample for sensitive analysis.

Conclusion In this work, we report on the development of a SH2 superbinder modified monolithic capillary microreactor and its application for the enrichment of phosphotyrosine peptides from complex peptide mixtures. To evaluate the feasibility of this strategy, we used protein standards as well as HeLa cell digests. The results showed that this microreactor enabled highly specific enrichment of phosphotyrosine 16


Page 16 of 28

Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


peptides. More than 700 pTyr sites were identified from phosphopeptide sample equivalent to only 100 µg HeLa cell digest. To the best of our knowledge, this is the first time that so many pTyr sites were identified from so small amount of samples by 1D RPLC-MS/MS analysis. Besides, the application to the analysis of a protein complex highlighted the great potential of this method toward small amount of clinical sample analysis. Recently, Abe et al. reported that significant improvement in analysis coverage could be achieved for the pTyr antibodies based method by optimization of phosphotyrosine enrichment and MS/MS parameters(9). We expect that the SH2 superbinder based method could also be further improved by optimization which will lead to deeper tyrosine phosphoproteome coverage.

Acknowledgments This work was supported, in part, by funds from the China State Key Basic Research Program Grants (2016YFA0501402), the National Natural Science Foundation of China (21605140, 21605140, 81600046, 21535008, 21235006). MY is a recipient of the National Science Fund of China for Distinguished Young Scholars (21525524). We also thank Prof. Shawn Li in University of Western (Canada) for providing the SH2 superbinder plasmids.

Competing financial interests: The authors declare no competing financial interest.

Supporting information The following files are available free of charge at ACS website http://pubs.acs.org: Figure S1. Scheme of immobilizing SH2 superbinder on the poly (glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column. Figure S2. Motif enrichment analysis for the pTyr sites identified from the Hela samples with different amounts of starting materials.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

Figure S3. Venn diagrams illustrating the unique pTyr sites found in SH2 microreactor method and SH2 sepharose method. Figure S4. Protein abundance distribution of the identified tyrosine phosphorylated proteins in SH2 microreactor enrichment method and SH2 sepharose beads enrichment method with 100 µg starting materials. Figure S5. Overlap of pTyr site identifications obtained from four SH2 microreactors in parallel. Table S1. Summary of the identifications obtained by the SH2 microreactor enrichment method and SH2 sepharose beads enrichment method.

References 1.

Rikova, K.; Guo, A.; Zeng, Q.; Possemato, A.; Yu, J.; Haack, H.; Nardone, J.; Lee, K.; Reeves, C.; Li, Y.;

Hu, Y.; Tan, Z.; Stokes, M.; Sullivan, L.; Mitchell, J.; Wetzel, R.; Macneill, J.; Ren, J. M.; Yuan, J.; Bakalarski, C. E.; Villen, J.; Kornhauser, J. M.; Smith, B.; Li, D.; Zhou, X.; Gygi, S. P.; Gu, T. L.; Polakiewicz, R. D.; Rush, J.; Comb, M. J., Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007, 131, (6), 1190-203. 2.

Blume-Jensen, P.; Hunter, T., Oncogenic kinase signalling. Nature 2001, 411, (6835), 355-65.

3.

Hunter, T., Tyrosine phosphorylation: thirty years and counting. Curr Opin Cell Biol 2009, 21, (2),

140-6. 4.

Lim, W. A.; Pawson, T., Phosphotyrosine signaling: evolving a new cellular communication system.

Cell 2010, 142, (5), 661-7. 5.

Talamonti, M. S.; Roh, M. S.; Curley, S. A.; Gallick, G. E., Increase in activity and level of pp60c-src

in progressive stages of human colorectal cancer. J Clin Invest 1993, 91, (1), 53-60. 6.

McKinley, E. T.; Liu, H.; McDonald, W. H.; Luo, W.; Zhao, P.; Coffey, R. J.; Hanks, S. K.; Manning, H.

C., Global phosphotyrosine proteomics identifies PKCdelta as a marker of responsiveness to Src inhibition in colorectal cancer. PLoS One 2013, 8, (11), e80207. 7.

Rush, J.; Moritz, A.; Lee, K. A.; Guo, A.; Goss, V. L.; Spek, E. J.; Zhang, H.; Zha, X. M.; Polakiewicz, R.

D.; Comb, M. J., Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 2005, 23, (1), 94-101. 8.

van der Mijn, J. C.; Labots, M.; Piersma, S. R.; Pham, T. V.; Knol, J. C.; Broxterman, H. J.; Verheul, H.

M.; Jimenez, C. R., Evaluation

of

different phospho-tyrosine antibodies

for label-free

phosphoproteomics. J Proteomics 2015, 127, (Pt B), 259-63. 9.

Abe, Y.; Nagano, M.; Tada, A.; Adachi, J.; Tomonaga, T., Deep Phosphotyrosine Proteomics by 18


Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Optimization of Phosphotyrosine Enrichment and MS/MS Parameters. J Proteome Res 2017, 16, (2), 1077-1086. 10. Sharma, K.; D'Souza, R. C.; Tyanova, S.; Schaab, C.; Wisniewski, J. R.; Cox, J.; Mann, M., Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling. Cell Rep 2014, 8, (5), 1583-94. 11. Yang, X.; Xia, Y., Selective enrichment and separation of phosphotyrosine peptides by thermosensitive molecularly imprinted polymers. J Sep Sci 2016, 39, (2), 419-26. 12. Xu, L.; Hu, Y.; Shen, F.; Li, Q.; Ren, X., Specific recognition of tyrosine-phosphorylated peptides by epitope imprinting of phenylphosphonic acid. J Chromatogr A 2013, 1293, 85-91. 13. Pawson, T.; Gish, G. D.; Nash, P., SH2 domains, interaction modules and cellular wiring. Trends Cell Biol 2001, 11, (12), 504-11. 14. Liu, B. A.; Jablonowski, K.; Raina, M.; Arce, M.; Pawson, T.; Nash, P. D., The human and mouse complement of SH2 domain proteins-establishing the boundaries of phosphotyrosine signaling. Mol Cell 2006, 22, (6), 851-68. 15. Machida, K.; Thompson, C. M.; Dierck, K.; Jablonowski, K.; Karkkainen, S.; Liu, B.; Zhang, H.; Nash, P. D.; Newman, D. K.; Nollau, P.; Pawson, T.; Renkema, G. H.; Saksela, K.; Schiller, M. R.; Shin, D. G.; Mayer, B. J., High-throughput phosphotyrosine profiling using SH2 domains. Mol Cell 2007, 26, (6), 899-915. 16. Kaneko, T.; Huang, H.; Cao, X.; Li, X.; Li, C.; Voss, C.; Sidhu, S. S.; Li, S. S., Superbinder SH2 domains act as antagonists of cell signaling. Sci Signal 2012, 5, (243), ra68. 17. Tong, J.; Cao, B.; Martyn, G. D.; Krieger, J. R.; Taylor, P.; Yates, B.; Sidhu, S. S.; Li, S. S.; Mao, X.; Moran, M. F., Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS. Proteomics 2017, 17, (6). 18. Bian, Y.; Li, L.; Dong, M.; Liu, X.; Kaneko, T.; Cheng, K.; Liu, H.; Voss, C.; Cao, X.; Wang, Y.; Litchfield, D.; Ye, M.; Li, S. S.; Zou, H., Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder. Nat Chem Biol 2016, 12, (11), 959-966. 19. Dong, M.; Bian, Y.; Wang, Y.; Dong, J.; Yao, Y.; Deng, Z.; Qin, H.; Zou, H.; Ye, M., Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis. Anal Chem 2017, 89, (17), 9307-9314. 20. Deng, Z.; Dong, M.; Wang, Y.; Dong, J.; Li, S. S.; Zou, H.; Ye, M., Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis. Anal Chem 2017, 89, (4), 2405-2410. 21. Amankwa, L. N.; Kuhr, W. G., Trypsin-Modified Fused-Silica Capillary Microreactor for Peptide-Mapping by Capillary Zone Electrophoresis. Analytical Chemistry 1992, 64, (14), 1610-1613. 22. Palm, A. K.; Novotny, M. V., Analytical characterization of a facile porous polymer monolithic trypsin microreactor enabling peptide mass mapping using mass spectrometry. Rapid Commun Mass Spectrom 2004, 18, (12), 1374-82. 23. Lin, L.; Liu, S.; Nie, Z.; Chen, Y.; Lei, C.; Wang, Z.; Yin, C.; Hu, H.; Huang, Y.; Yao, S., Automatic and integrated micro-enzyme assay (AImuEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column. Anal Chem 2015, 87, (8), 4552-9. 24. Zhang, S.; Yuan, H.; Zhao, B.; Zhou, Y.; Jiang, H.; Zhang, L.; Liang, Z.; Zhang, Y., Integrated platform with a combination of online digestion and (18)O labeling for proteome quantification via an immobilized trypsin microreactor. Analyst 2015, 140, (15), 5227-34. 25. Yin, J.; Xu, T.; Zhang, N.; Wang, H., Three-Enzyme Cascade Bioreactor for Rapid Digestion of 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Genomic DNA into Single Nucleosides. Anal Chem 2016, 88, (15), 7730-7. 26. Feng, S.; Pan, C.; Jiang, X.; Xu, S.; Zhou, H.; Ye, M.; Zou, H., Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis. Proteomics 2007, 7, (3), 351-60. 27. Palm, A. K.; Novotny, M. V., A monolithic PNGase F enzyme microreactor enabling glycan mass mapping of glycoproteins by mass spectrometry. Rapid Commun Mass Spectrom 2005, 19, (12), 1730-8. 28. Peterson, D. S.; Rohr, T.; Svec, F.; Fréchet, J. M. J., Enzymatic Microreactor-on-a-Chip: Protein Mapping Using Trypsin Immobilized on Porous Polymer Monoliths Molded in Channels of Microfluidic Devices. Analytical Chemistry 2002, 74, (16), 4081-4088. 29. Wu, R.; Zou, H.; Ye, M.; Lei, Z.; Ni, J., Separation of basic, acidic and neutral compounds by capillary electrochromatography using uncharged monolithic capillary columns modified with anionic and cationic surfactants. Electrophoresis 2001, 22, (3), 544-551. 30. Ye, M.; Hu, S.; Schoenherr, R. M.; Dovichi, N. J., On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection. Electrophoresis 2004, 25, (9), 1319-26. 31. Huyer, G.; Liu, S.; Kelly, J.; Moffat, J.; Payette, P.; Kennedy, B.; Tsaprailis, G.; Gresser, M. J.; Ramachandran, C., Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate. J Biol Chem 1997, 272, (2), 843-51. 32. Yao, Y.; Dong, J.; Dong, M.; Liu, F.; Wang, Y.; Mao, J.; Ye, M.; Zou, H., An immobilized titanium (IV) ion affinity chromatography adsorbent for solid phase extraction of phosphopeptides for phosphoproteome analysis. J Chromatogr A 2017, 1498, 22-28. 33. Cox, J.; Mann, M., MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 2008, 26, (12), 1367-72. 34. Doerks, T.; Copley, R. R.; Schultz, J.; Ponting, C. P.; Bork, P., Systematic identification of novel protein domain families associated with nuclear functions. Genome Res 2002, 12, (1), 47-56. 35. Petro, M.; Svec, F.; Fréchet, J. M. J., Immobilization of trypsin onto “molded” macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) rods and use of the conjugates as bioreactors and for affinity chromatography. Biotechnology and Bioengineering 2000, 49, (4), 355-363. 36. Feng, S.; Ye, M.; Jiang, X.; Jin, W.; Zou, H., Coupling the immobilized trypsin microreactor of monolithic capillary with muRPLC-MS/MS for shotgun proteome analysis. J Proteome Res 2006, 5, (2), 422-8. 37. Wang, M.; Weiss, M.; Simonovic, M.; Haertinger, G.; Schrimpf, S. P.; Hengartner, M. O.; von Mering, C., PaxDb, a Database of Protein Abundance Averages Across All Three Domains of Life. Molecular & Cellular Proteomics 2012, 11, (8), 492-500. 38. Di Palma, S.; Zoumaro-Djayoon, A.; Peng, M.; Post, H.; Preisinger, C.; Munoz, J.; Heck, A. J., Finding the same needles in the haystack? A comparison of phosphotyrosine peptides enriched by immuno-affinity precipitation and metal-based affinity chromatography. J Proteomics 2013, 91, 331-7. 39. Luzi, L.; Confalonieri, S.; Di Fiore, P. P.; Pelicci, P. G., Evolution of Shc functions from nematode to human. Curr Opin Genet Dev 2000, 10, (6), 668-74. 40. Ong, S. H.; Dilworth, S.; Hauck-Schmalenberger, I.; Pawson, T.; Kiefer, F., ShcA and Grb2 mediate polyoma middle T antigen-induced endothelial transformation and Gab1 tyrosine phosphorylation. EMBO J 2001, 20, (22), 6327-36. 20


Page 20 of 28

Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


41. Ursini-Siegel, J.; Hardy, W. R.; Zuo, D.; Lam, S. H.; Sanguin-Gendreau, V.; Cardiff, R. D.; Pawson, T.; Muller, W. J., ShcA signalling is essential for tumour progression in mouse models of human breast cancer. EMBO J 2008, 27, (6), 910-20. 42. Zheng, Y.; Zhang, C.; Croucher, D. R.; Soliman, M. A.; St-Denis, N.; Pasculescu, A.; Taylor, L.; Tate, S. A.; Hardy, W. R.; Colwill, K.; Dai, A. Y.; Bagshaw, R.; Dennis, J. W.; Gingras, A. C.; Daly, R. J.; Pawson, T., Temporal regulation of EGF signalling networks by the scaffold protein Shc1. Nature 2013, 499, (7457), 166-71. 43. Dyson, M. R.; Zheng, Y.; Zhang, C.; Colwill, K.; Pershad, K.; Kay, B. K.; Pawson, T.; McCafferty, J., Mapping protein interactions by combining antibody affinity maturation and mass spectrometry. Anal Biochem 2011, 417, (1), 25-35. 44. Hornbeck, P. V.; Zhang, B.; Murray, B.; Kornhauser, J. M.; Latham, V.; Skrzypek, E., PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 2015, 43, (Database issue), D512-20.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Captions Figure 1. Scanning electron micrographs of SH2 superbinder modified monolithic capillary column with 200 µm i.d. (a) Cross section of the affinity monolithic column, (b) structure of the affinity monolith inside the capillary. Figure 2. MALDI-TOF MS spectrum of the peptide mixture of pY, α-casein and β-casein with molar ratio of (a) 1:100:100, direct analysis, (b) 1:100:100, after enrichment by SH2 microreactor, (c) 1:500:500, after enrichment by SH2 microreactor, (d) 1:1000:1000, after enrichment by SH2 microreactor. Figure 3. MALDI-TOF MS spectrum of the peptide mixture of pY, α-casein and β-casein with molar ratio of 1:1:1, (a) direct analysis, after enrichment by SH2 microreactor with the sample amount of (b) 5 fmol, (c) 1 fmol, (d) 0.1 fmol. Figure 4. Comparison of pTyr identifications obtained by the SH2 microreactor enrichment method and SH2 sepharose beads enrichment method. Different amounts of starting materials were respectively enriched by the indicated two methods. Two replicate LC–MS/MS runs were performed, and the results were combined. All sites have a Localization Probability ≥ 0.75 and Score Difference ≥ 5. Figure 5. Summary of the pTyr sites identified from Shc1 complex before and after enrichment by the SH2 microreactor. Two replicate LC–MS/MS runs were performed, and the results were combined. All sites have a Localization Probability ≥ 0.75 and Score Difference ≥ 5, (a) the number of identified pTyr sites belonging to Shc1 complex, (b) all identified pTyr sites in the Shc1 complex. pTyr sites only identified before enrichment are shown by a blue star, pTyr sites only identified after enrichment are shown by a red star, and pTyr sites identified both before and after enrichment are shown by a black star, (c) the number of identification records with proteomic discovery-mode mass spectrometry in PhosphositePlus dataset for the 16 pTyr sites identified on Shc1 complex.

22


Page 22 of 28

Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2.

24


Page 24 of 28

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4

26


Page 26 of 28

Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure5

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

for TOC only

28


Page 28 of 28

SH2 Superbinder Modified Monolithic Capillary Column for the

Recommend Documents