Advancing the analysis of less specific proteolytic digests via nLC–ESI-QoTOF-MS/MS

Abstract Although less specific proteases have been established in proteomics, resulting peptides are often disadvantaged in mass spectrometric analysis compared to tryptic peptides. This is due to the high number of theoretically possible peptides as well as their relatively inefficient ionization and fragmentation. One possibility to improve the performance of less specific digests is to employ amine reactive, basic labels such as tandem mass tags (TMT) for the N-terminal derivatization of peptides. This study uses elastase and thermolysin digestion of cytosolic extracts with and without the use of TMT labeling for analysis via nanoflow high-performance liquid chromatography (nLC) coupled to an electrospray ionization quadrupole orthogonal acceleration time-of-flight (ESI-QoTOF) mass spectrometer. Differences between tryptic and less specific digests and their behavior during chromatographic separation and tandem mass spectrometric (MS/MS) analysis are highlighted. Furthermore, this work shows how the tandem mass spectrometric analysis of less specific digests can be optimized with reference to peptide ionization and fragmentation. The effect of TMT labeling on the number and characteristics of identified peptides as well as peptide fragmentation behavior is examined. Taking all these factors into consideration, this work constitutes an optimized strategy for the analysis of less specific digests via nLC–ESI-QoTOF-MS/MS.