ABSTRACT We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr

superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified

∼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2

domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation.

characterization of the tyrosine phosphoproteome under normal or pathological conditions. Access through your institution Buy or subscribe This is a preview of subscription content, access

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PHOSPHATASES BY MASS SPECTROMETRY Article 13 September 2021 A DATA-INDEPENDENT ACQUISITION-BASED GLOBAL PHOSPHOPROTEOMICS SYSTEM ENABLES DEEP PROFILING Article Open access 05 May 2021 THE

INTRINSIC SUBSTRATE SPECIFICITY OF THE HUMAN TYROSINE KINOME Article Open access 08 May 2024 ACCESSION CODES ACCESSIONS PROTEIN DATA BANK * 1EEO * 4F5B REFERENCES * Sharma, K. et al.

Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling. _Cell Rep._ 8, 1583–1594 (2014). Article  CAS  PubMed  Google Scholar  * Manning, G.,

Whyte, D.B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. _Science_ 298, 1912–1934 (2002). Article  CAS  PubMed  Google Scholar  *

Wilhelm, M. et al. Mass-spectrometry-based draft of the human proteome. _Nature_ 509, 582–587 (2014). Article  CAS  PubMed  Google Scholar  * Kim, M.S. et al. A draft map of the human

proteome. _Nature_ 509, 575–581 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, L. et al. Integrated omic analysis of lung cancer reveals metabolism proteome signatures

with prognostic impact. _Nat. Commun._ 5, 5469 (2014). Article  CAS  PubMed  Google Scholar  * Zhou, H. et al. Robust phosphoproteome enrichment using monodisperse microsphere-based

immobilized titanium (IV) ion affinity chromatography. _Nat. Protoc._ 8, 461–480 (2013). Article  CAS  PubMed  Google Scholar  * de Graaf, E.L., Giansanti, P., Altelaar, A.F. & Heck,

A.J. Single-step enrichment by Ti4+-IMAC and label-free quantitation enables in-depth monitoring of phosphorylation dynamics with high reproducibility and temporal resolution. _Mol. Cell.

Proteomics_ 13, 2426–2434 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rikova, K. et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung

cancer. _Cell_ 131, 1190–1203 (2007). Article  CAS  PubMed  Google Scholar  * Zhang, Y. et al. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth

factor receptor signaling network reveals dynamic modules. _Mol. Cell. Proteomics_ 4, 1240–1250 (2005). Article  CAS  PubMed  Google Scholar  * Tinti, M. et al. The 4G10, pY20 and p-TYR-100

antibody specificity: profiling by peptide microarrays. _Nat. Biotechnol._ 29, 571–577 (2012). CAS  Google Scholar  * Jones, R.B., Gordus, A., Krall, J.A. & MacBeath, G. A quantitative

protein interaction network for the ErbB receptors using protein microarrays. _Nature_ 439, 168–174 (2006). Article  CAS  PubMed  Google Scholar  * Kaneko, T. et al. Superbinder SH2 domains

act as antagonists of cell signaling. _Sci. Signal._ 5, ra68 (2012). Article  PubMed  CAS  Google Scholar  * Rush, J. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer

cells. _Nat. Biotechnol._ 23, 94–101 (2005). Article  CAS  PubMed  Google Scholar  * Findlay, G.M. et al. Interaction domains of Sos1/Grb2 are finely tuned for cooperative control of

embryonic stem cell fate. _Cell_ 152, 1008–1020 (2013). Article  CAS  PubMed  Google Scholar  * Huyer, G. et al. Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and

pervanadate. _J. Biol. Chem._ 272, 843–851 (1997). Article  CAS  PubMed  Google Scholar  * Huang, H. et al. Defining the specificity space of the human SRC homology 2 domain. _Mol. Cell.

Proteomics_ 7, 768–784 (2008). Article  CAS  PubMed  Google Scholar  * Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A.J. Multiplex peptide stable isotope dimethyl

labeling for quantitative proteomics. _Nat. Protoc._ 4, 484–494 (2009). Article  CAS  PubMed  Google Scholar  * Subik, K. et al. The Expression Patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67

and AR by Immunohistochemical Analysis in Breast Cancer Cell Lines. _Breast Cancer (Auckl.)_ 4, 35–41 (2010). PubMed Central  Google Scholar  * Matlock, M.K., Holehouse, A.S. & Naegle,

K.M. ProteomeScout: a repository and analysis resource for post-translational modifications and proteins. _Nucleic Acids Res._ 43, D521–D530 (2015). Article  CAS  PubMed  Google Scholar  *

Hornbeck, P.V. et al. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse.

_Nucleic Acids Res._ 40, D261–D270 (2012). Article  CAS  PubMed  Google Scholar  * Lu, C.T. et al. DbPTM 3.0: an informative resource for investigating substrate site specificity and

functional association of protein post-translational modifications. _Nucleic Acids Res._ 41, D295–D305 (2013). Article  CAS  PubMed  Google Scholar  * UniProt Consortium. Activities at the

Universal Protein Resource (UniProt). _Nucleic Acids Res._ 42, D191–D198 (2014). * Geiger, T., Wehner, A., Schaab, C., Cox, J. & Mann, M. Comparative proteomic analysis of eleven common

cell lines reveals ubiquitous but varying expression of most proteins. _Mol. Cell. Proteomics_ 11, M111.014050 (2012). Article  PubMed  PubMed Central  CAS  Google Scholar  * Stokes, M.P. et

al. Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis. _Proteomes_ 3, 160–183 (2015). Article  PubMed 

PubMed Central  Google Scholar  * Hubbard, S.R., Mohammadi, M. & Schlessinger, J. Autoregulatory mechanisms in protein-tyrosine kinases. _J. Biol. Chem._ 273, 11987–11990 (1998). Article

  CAS  PubMed  Google Scholar  * Qian, X. et al. The Tensin-3 protein, including its SH2 domain, is phosphorylated by Src and contributes to tumorigenesis and metastasis. _Cancer Cell_ 16,

246–258 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Jin, L.L. et al. Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and

specificity. _Mol. Cell. Proteomics_ 14, 695–706 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Andersen, J.N. et al. Structural and evolutionary relationships among protein

tyrosine phosphatase domains. _Mol. Cell. Biol._ 21, 7117–7136 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mitra, S., Beach, C., Feng, G.S. & Plattner, R. SHP-2 is a

novel target of Abl kinases during cell proliferation. _J. Cell Sci._ 121, 3335–3346 (2008). Article  CAS  PubMed  Google Scholar  * Feng, J. et al. Activation of Jak2 catalytic activity

requires phosphorylation of Y1007 in the kinase activation loop. _Mol. Cell. Biol._ 17, 2497–2501 (1997). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, J., Billingsley,

M.L., Kincaid, R.L. & Siraganian, R.P. Phosphorylation of Syk activation loop tyrosines is essential for Syk function. An in vivo study using a specific anti-Syk activation loop

phosphotyrosine antibody. _J. Biol. Chem._ 275, 35442–35447 (2000). Article  CAS  PubMed  Google Scholar  * Schindler, T. et al. Structural mechanism for STI-571 inhibition of abelson

tyrosine kinase. _Science_ 289, 1938–1942 (2000). Article  CAS  PubMed  Google Scholar  * Huse, M. & Kuriyan, J. The conformational plasticity of protein kinases. _Cell_ 109, 275–282

(2002). Article  CAS  PubMed  Google Scholar  * Xie, Y., Pendergast, A.M. & Hung, M.C. Dominant-negative mutants of Grb2 induced reversal of the transformed phenotypes caused by the

point mutation-activated rat HER-2/Neu. _J. Biol. Chem._ 270, 30717–30724 (1995). Article  CAS  PubMed  Google Scholar  * Sepp-Lorenzino, L. et al. Signal transduction pathways induced by

heregulin in MDA-MB-453 breast cancer cells. _Oncogene_ 12, 1679–1687 (1996). CAS  PubMed  Google Scholar  * Dey, B.R., Frick, K., Lopaczynski, W., Nissley, S.P. & Furlanetto, R.W.

Evidence for the direct interaction of the insulin-like growth factor I receptor with IRS-1, Shc, and Grb10. _Mol. Endocrinol._ 10, 631–641 (1996). CAS  PubMed  Google Scholar  *

Tartare-Deckert, S., Sawka-Verhelle, D., Murdaca, J. & Van Obberghen, E. Evidence for a differential interaction of SHC and the insulin receptor substrate-1 (IRS-1) with the insulin-like

growth factor-I (IGF-I) receptor in the yeast two-hybrid system. _J. Biol. Chem._ 270, 23456–23460 (1995). Article  CAS  PubMed  Google Scholar  * Barretina, J. et al. The Cancer Cell Line

Encyclopedia enables predictive modelling of anticancer drug sensitivity. _Nature_ 483, 603–607 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Takeuchi, K. & Ito, F.

Receptor tyrosine kinases and targeted cancer therapeutics. _Biol. Pharm. Bull._ 34, 1774–1780 (2011). Article  CAS  PubMed  Google Scholar  * Levitzki, A. Tyrosine kinase inhibitors: views

of selectivity, sensitivity, and clinical performance. _Annu. Rev. Pharmacol. Toxicol._ 53, 161–185 (2013). Article  CAS  PubMed  Google Scholar  * Zaman, N. et al. Signaling network

assessment of mutations and copy number variations predict breast cancer subtype-specific drug targets. _Cell Rep._ 5, 216–223 (2013). Article  CAS  PubMed  Google Scholar  * Esteva, F.J.,

Yu, D., Hung, M.C. & Hortobagyi, G.N. Molecular predictors of response to trastuzumab and lapatinib in breast cancer. _Nat. Rev. Clin. Oncol._ 7, 98–107 (2010). Article  CAS  PubMed 

Google Scholar  * Sabbatini, P. et al. GSK1838705A inhibits the insulin-like growth factor-1 receptor and anaplastic lymphoma kinase and shows antitumor activity in experimental models of

human cancers. _Mol. Cancer Ther._ 8, 2811–2820 (2009). Article  CAS  PubMed  Google Scholar  * Kim, H.G. et al. Discovery of a potent and selective DDR1 receptor tyrosine kinase inhibitor.

_ACS Chem. Biol._ 8, 2145–2150 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang, S. et al. Combating trastuzumab resistance by targeting SRC, a common node downstream of

multiple resistance pathways. _Nat. Med._ 17, 461–469 (2011). Article  PubMed  CAS  Google Scholar  * Lee, M.J. et al. Sequential application of anticancer drugs enhances cell death by

rewiring apoptotic signaling networks. _Cell_ 149, 780–794 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hochgräfe, F. et al. Tyrosine phosphorylation profiling reveals the

signaling network characteristics of Basal breast cancer cells. _Cancer Res._ 70, 9391–9401 (2010). Article  PubMed  CAS  Google Scholar  * Lemmon, M.A. & Schlessinger, J. Cell

signaling by receptor tyrosine kinases. _Cell_ 141, 1117–1134 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Krause, D.S. & Van Etten, R.A. Tyrosine kinases as targets

for cancer therapy. _N. Engl. J. Med._ 353, 172–187 (2005). Article  CAS  PubMed  Google Scholar  * Debnath, J., Muthuswamy, S.K. & Brugge, J.S. Morphogenesis and oncogenesis of MCF-10A

mammary epithelial acini grown in three-dimensional basement membrane cultures. _Methods_ 30, 256–268 (2003). Article  CAS  PubMed  Google Scholar  * Boersema, P.J. et al. In-depth

qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling. _Mol. Cell.

Proteomics_ 9, 84–99 (2010). Article  CAS  PubMed  Google Scholar  * Zhou, H. et al. Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for

phosphoproteome analysis. _J. Proteome Res._ 7, 3957–3967 (2008). Article  CAS  PubMed  Google Scholar  * Wang, F. et al. A fully automated system with online sample loading, isotope

dimethyl labeling and multidimensional separation for high-throughput quantitative proteome analysis. _Anal. Chem._ 82, 3007–3015 (2010). Article  CAS  PubMed  Google Scholar  * Olsen, J.V.

et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. _Cell_ 127, 635–648 (2006). Article  CAS  PubMed  Google Scholar  Download references

ACKNOWLEDGEMENTS This work was supported, in part, by funds from the China State Key Basic Research Program Grants (2012CB910101 and 2016YFA0501402 to M.Y. and 2013CB911202 to H.Z.), the

National Natural Science Foundation of China (21235006, 21321064 and 21535008 to H.Z., and 81361128015 to M.Y. and S.S.-C.L.), the Canadian Cancer Society (to S.S.-C.L.), the Canadian

Institute of Health Research (to S.S.-C.L. and M.Y.) and the Ontario Research Fund (to S.S.-C.L.). L.L. is a recipient of “Distinguished Expert of Overseas Tai Shan Scholar”. M.Y. is a

recipient of the National Science Fund of China for Distinguished Young Scholars (21525524). S.S.-C.L. holds a Canadian Research Chair in Functional Genomics and Cellular Proteomics. AUTHOR

INFORMATION Author notes * Yangyang Bian, Lei Li and Mingming Dong: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Key Laboratory of Separation Sciences for

Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China Yangyang Bian, Mingming Dong, Kai Cheng,

 Yan Wang, Mingliang Ye & Hanfa Zou * University of Chinese Academy of Sciences, Beijing, China Yangyang Bian & Mingming Dong * Department of Biochemistry, Schulich School of

Medicine and Dentistry, Western University, London, Ontario, Canada., Lei Li, Xuguang Liu, Tomonori Kaneko, Huadong Liu, Courtney Voss, Xuan Cao, David Litchfield & Shawn S-C Li * School

of Basic Medical Sciences, Qingdao University, Qingdao, China Lei Li * Department of Oncology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada., David

Litchfield & Shawn S-C Li * The Children's Health Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada., Shawn S-C Li Authors

* Yangyang Bian View author publications You can also search for this author inPubMed Google Scholar * Lei Li View author publications You can also search for this author inPubMed Google

Scholar * Mingming Dong View author publications You can also search for this author inPubMed Google Scholar * Xuguang Liu View author publications You can also search for this author

inPubMed Google Scholar * Tomonori Kaneko View author publications You can also search for this author inPubMed Google Scholar * Kai Cheng View author publications You can also search for

this author inPubMed Google Scholar * Huadong Liu View author publications You can also search for this author inPubMed Google Scholar * Courtney Voss View author publications You can also

search for this author inPubMed Google Scholar * Xuan Cao View author publications You can also search for this author inPubMed Google Scholar * Yan Wang View author publications You can

also search for this author inPubMed Google Scholar * David Litchfield View author publications You can also search for this author inPubMed Google Scholar * Mingliang Ye View author

publications You can also search for this author inPubMed Google Scholar * Shawn S-C Li View author publications You can also search for this author inPubMed Google Scholar * Hanfa Zou View

author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.S.-C.L., H.Z., M.Y. and M.D. conceived and designed the project. Y.B. and M.D. carried out the

pTyr enrichment experiments and MS analysis under the supervision of H.Z., M.Y. and S.S.-C.L. L.L., M.Y., Y.B., M.D., K.C., T.K. and S.S.-C.L. analyzed the data. X.L., Y.W. and X.C.

performed the cellular and biochemical experiments with input from S.S.-C.L. and D.L. C.V. and H.L. prepared the superbinder reagents. S.S.-C.L., L.L. and M.Y. wrote the manuscript with

input from H.Z., M.D., Y.B. and D.L. CORRESPONDING AUTHORS Correspondence to Mingliang Ye, Shawn S-C Li or Hanfa Zou. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–19. (PDF 9135 kb) SUPPLEMENTARY

DATASET Supplementary Datasets 1–8. (XLSX 41064 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Bian, Y., Li, L., Dong, M. _et al._ Ultra-deep

tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder. _Nat Chem Biol_ 12, 959–966 (2016). https://doi.org/10.1038/nchembio.2178 Download citation * Received: 18 August 2015 *

Accepted: 11 July 2016 * Published: 19 September 2016 * Issue Date: November 2016 * DOI: https://doi.org/10.1038/nchembio.2178 SHARE THIS ARTICLE Anyone you share the following link with

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