~(14)N15N DXMSMS Match program for the automated analysis of LC/ESI-MS/MS crosslinking data from experiments using 15N metabolically labeled proteins

摘要

Crosslinking mass spectrometric applications for the study of proteins and protein complexes benefit from using 15N metabolically labeled proteins. Peptides, derived from crosslinked 14N and 15N proteins (used in a 1:1 molar ratio), exhibit specific mass spectrometric signatures of doublets of peaks, reflecting the number of nitrogen atoms in the peptides. This can be used as an additional search criterion for assignment of the crosslinks.Here, we describe the further development of our ICC-CLASS software suite which is designed for automatic analysis of mass spectrometric crosslinking data, by the addition of the 14N15N DXMSMS Match program. The program is designed to assist in distinguishing inter- from intra-molecular crosslinks at the interface of homodimers in protein aggregation studies. The program takes into account the number of nitrogen atoms present in 14N15N-labeled crosslinked peptides and uses it as an additional parameter for the identification of crosslinks based on both the MS and MS/MS spectra. This greatly increases the confidence of the assignments, and this approach can be successfully used in other types of complicated crosslinking experiments, such as those with non-specific crosslinking sites, non-specific digestion, zero-length crosslinking, or crosslinking with unknown reaction mechanisms, by facilitating the use of 15N metabolically labeled proteins.