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      Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

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          Abstract

          Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

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          Author and article information

          Journal
          Anal. Chem.
          Analytical chemistry
          American Chemical Society (ACS)
          1520-6882
          0003-2700
          Feb 21 2017
          : 89
          : 4
          Affiliations
          [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 1000491, China.
          [3 ] Departments of Biochemistry, Oncology and the Children's Health Research Institute, Schulich School of Medicine and Dentistry, Western University , London, Ontario N6A 5C1, Canada.
          Article
          10.1021/acs.analchem.6b04288
          28192900

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