Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis

Abstract

Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction. However, its analysis at the proteome level remains challenging. The conventional immunopurification (IP) approach using antibodies specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. Our recent study indicated that SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we presented an efficient SH2 superbinder based workflow for the sensitive analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr peptides than the previous method. Its excellent performance was demonstrated by the analysis of a variety of different samples. For the highly tyrosine phosphorylated sample, for example, pervanadate-treated Jurkat T cells, it identified over 1800 high confident pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells, where the pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of proteins, which was 31% more than that obtained by the antibody-based method. For the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and cost-effective approach and would have wide applications in the study of the regulatory role of tyrosine phosphorylation in diverse physiological and pathological processes.

Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials

“…This technique, however, is limited by the biotin-pYEEI used to elute pTyr from the SH2 super-binder, as it must be removed from the sample before LC-MS/MS analysis. The additional purification step results in significant sample loss [103]. The low recovery of these biological enrichment methods limits their applications to untreated cells or tissue samples with much lower pTyr levels.…”

Challenges and Advances in the Fabrication of Monolithic Bioseparation Materials and their Applications in Proteomics Research

“…Selective enrichment of phosphorylated targets was assessed, and the results confirmed that the imprinted monoliths could be useful as trapping media in affinity‐based phosphoproteomics. Our group selected a Src homology 2 (SH2)‐domain‐derived phosphotyrosine (pTyr) superbinder as an affinity ligand for specific enrichment of pTyr. About 20 000 phosphotyrosyl peptides and more than 10 000 pTyr sites could be recognized by LC–MS.…”

Aptamer‐SH2 superbinder‐based targeted therapy for pancreatic ductal adenocarcinoma

“…The SH2 domain with triple mutants, called SH2 superbinder herein, was identified to have stronger affinity to pY residues than the natural counterpart and might efficiently capture diverse pY peptides. [22][23][24][25] Especially, the minor difference of pY sequence context was verified to have no obvious effect on the binding preference under condition of excess SH2 superbinders. 25 After introduced into tumor cells, SH2 superbinder would displace the endogenous SH2 domain-containing proteins and block a large number of pY signaling pathways.…”

“…Generally, the natural SH2 domains present moderate affinity to pY residues and have different pY sequence binding preferences. The SH2 domain with triple mutants, called SH2 superbinder herein, was identified to have stronger affinity to pY residues than the natural counterpart and might efficiently capture diverse pY peptides 22–25 . Especially, the minor difference of pY sequence context was verified to have no obvious effect on the binding preference under condition of excess SH2 superbinders 25 .…”