Quantitative Analysis of Protein Covalent Labeling Mass Spectrometry Data in the Mass Spec Studio

Covalent
labeling with mass spectrometry (CL-MS) provides a direct
measure of the chemical and structural features of proteins with the
potential for resolution at the amino-acid level. Unfortunately, most
applications of CL-MS are limited to narrowly defined differential
analyses, where small numbers of residues are compared between two
or more protein states. Extending the utility of high-resolution CL-MS
for structure-based applications requires more robust computational
routines and the development of methodology capable of reporting of
labeling yield accurately. Here, we provide a substantial improvement
in the analysis of CL-MS data with the development of an extended
plug-in built within the Mass Spec Studio development framework (MSS-CLEAN).
All elements of data analysisfrom database search to site-resolved
and normalized labeling outputare accommodated, as illustrated
through the nonselective labeling of the human kinesin Eg5 with photoconverted
3,3′-azibutan-1-ol. In developing the new features within the
CL-MS plug-in, we identified additional complexities associated with
the application of CL reagents, arising primarily from digestion-induced
bias in yield measurements and ambiguities in site localization. A
strategy is presented involving the use of redundant site labeling
data from overlapping peptides, the imputation of missing data, and
a normalization routine to determine relative protection factors.
These elements together provide for a robust structural interpretation
of CL-MS/MS data while minimizing the over-reporting of labeling site
resolution. Finally, to minimize bias, we recommend that digestion
strategies for the generation of useful overlapping peptides involve
the application of complementary enzymes that drive digestion to completion.