Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale

Described
here is a chemo-selective enrichment strategy, termed
the semitryptic peptide enrichment strategy for proteolysis procedures
(STEPP), to isolate the semitryptic peptides generated in mass spectrometry-based
proteome-wide applications of limited proteolysis methods. The strategy
involves reacting the ε-amino groups of lysine side chains and
any N-termini created in the limited proteolysis reaction with isobaric
mass tags. A subsequent digestion of the sample with trypsin and the
chemo-selective reaction of the newly exposed N-termini of the tryptic
peptides with N-hydroxysuccinimide (NHS)-activated
agarose resin removes the tryptic peptides from solution, leaving
only the semitryptic peptides with one nontryptic cleavage site generated
in the limited proteolysis reaction for subsequent LC–MS/MS
analysis. As part of this work, the STEPP technique is interfaced
with two different proteolysis methods, including the pulse proteolysis
(PP) and limited proteolysis (LiP) methods. The STEPP-PP workflow
is evaluated in two proof-of-principle experiments involving the proteins
in a yeast cell lysate and two well-studied drugs, cyclosporin A and
geldanamycin. The STEPP-LiP workflow is evaluated in a proof-of-principle
experiment involving the proteins in two cell culture models of human
breast cancer, MCF-7 and MCF-10A cell lines. The STEPP protocol increased
the number of semitryptic peptides detected in the LiP and PP experiments
by 5- to 10-fold. The STEPP protocol not only increases the proteomic
coverage, but also increases the amount of structural information
that can be gleaned from limited proteolysis experiments. Moreover,
the protocol also enables the quantitative determination of ligand
binding affinities.