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Fast track to a phosphoprotein sketch – MALDI‐TOF characterization of TLC‐based tryptic phosphopeptide maps at femtomolar detection sensitivity
Author(s) -
Kochin Vitaly,
Imanishi Susumu Y.,
Eriksson John E.
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200600457
Subject(s) - phosphopeptide , phosphoproteomics , chromatography , chemistry , phosphoprotein , mass spectrometry , phosphorylation , biochemistry , protein phosphorylation , protein kinase a
Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given 32 P‐labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2‐D phosphopeptide maps (2‐D‐PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI‐TOF MS analysis of phosphopeptides extracted from 2‐D‐PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro 32 P‐labeled proteins.