A Cautionary Tale on the Inclusion of Variable Posttranslational Modifications in Database-Dependent Searches of Mass...

Mass spectrometry-based proteomics allows in principle the identification of unknown target proteins of posttranslational modifications and the sites of attachment. Including a variety of posttranslational modifications in database-dependent searches of high-throughput mass spectrometry data holds the promise to gain spectrum assignments to modified peptides, thereby increasing the number of assigned spectra, and to identify potentially interesting modification events. However, these potential benefits come for the price of an increased search space, which can lead to reduced scores, increased score thresholds, and erroneous peptide spectrum matches. We have assessed here the advantages and disadvantages of including the variable posttranslational modifications methionine oxidation, protein N-terminal acetylation, cysteine carbamidomethylation, transformation of N-terminal glutamine to pyroglutamic acid (Gln→pyro-Glu), and deamidation of asparagine and glutamine. Based on calculations of local false discovery rates and comparisons to known features of the respective modifications, we recommend for searches of samples that were not enriched for specific posttranslational modifications to only include methionine oxidation, protein N-terminal acetylation, and peptide N-terminal Gln→pyro-Glu as variable modifications. The principle of the validation strategy adopted here can also be applied for assessing the inclusion of posttranslational modifications for differently prepared samples, or for additional modifications. In addition, we have reassessed the special properties of the ubiquitin footprint, which is the remainder of ubiquitin moieties attached to lysines after tryptic digest. We show here that the ubiquitin footprint often breaks off as neutral loss and that it can be distinguished from dicarbamidomethylation events.