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J Proteomics. 2014 Sep 23;109:104-10. doi: 10.1016/j.jprot.2014.06.014. Epub 2014 Jun 25.

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

Author information

1
Genome British Columbia Proteomics Centre, University of Victoria, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z 7X8, Canada. Electronic address: jenya@proteincentre.com.
2
Genome British Columbia Proteomics Centre, University of Victoria, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z 7X8, Canada.
3
Genome British Columbia Proteomics Centre, University of Victoria, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z 7X8, Canada; Department of Biochemistry & Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.

Abstract

Crosslinking mass spectrometric applications for the study of proteins and protein complexes benefit from using (15)N metabolically labeled proteins. Peptides, derived from crosslinked (14)N and (15)N proteins (used in a 1:1molar 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 (14)N(15)N 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 (14)N(15)N-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 (15)N metabolically labeled proteins.

BIOLOGICAL SIGNIFICANCE:

The new (14)N(15)N DXMSMS Match software program is a practical tool for the efficient assignment of crosslinks from LC-MS/MS experiments using an equimolar mixture of non-labeled and (15)N metabolically labeled proteins. It greatly facilitates automated data analysis from complicated crosslinking experiments, such as those using zero-length crosslinkers and those involving only a few crosslinking and digestion site restrictions.

KEYWORDS:

(15)N metabolic labeling; Crosslinking; Mass spectrometry

PMID:
24972318
DOI:
10.1016/j.jprot.2014.06.014
[Indexed for MEDLINE]

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