ac7b02078_si_003.xlsx (954.07 kB)
Download fileSensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis
dataset
posted on 2017-08-10, 00:00 authored by Mingming Dong, Yangyang Bian, Yan Wang, Jing Dong, Yating Yao, Zhenzhen Deng, Hongqiang Qin, Hanfa Zou, Mingliang YeAlbeit 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.