ISPTM: An Iterative Search Algorithm for Systematic Identification of Post-translational Modifications from Complex Proteome Mixtures

Identifying protein post-translational
modifications (PTMs) from
tandem mass spectrometry data of complex proteome mixtures is a highly
challenging task. Here we present a new strategy, named iterative
search for identifying PTMs (ISPTM), for tackling this challenge.
The ISPTM approach consists of a basic search with no variable modification,
followed by iterative searches of many PTMs using a small number of
them (usually two) in each search. The performance of the ISPTM approach
was evaluated on mixtures of 70 synthetic peptides with known modifications,
on an 18-protein standard mixture with unknown modifications and on
real, complex biological samples of mouse nuclear matrix proteins
with unknown modifications. ISPTM revealed that many chemical PTMs
were introduced by urea and iodoacetamide during sample preparation
and many biological PTMs, including dimethylation of arginine and
lysine, were significantly activated by Adriamycin treatment in nuclear
matrix associated proteins. ISPTM increased the MS/MS spectral identification
rate substantially, displayed significantly better sensitivity for
systematic PTM identification compared with that of the conventional
all-in-one search approach, and offered PTM identification results
that were complementary to InsPecT and MODa, both of which are established
PTM identification algorithms. In summary, ISPTM is a new and powerful
tool for unbiased identification of many different PTMs with high
confidence from complex proteome mixtures.