Use of a combination of isotopically coded cross-linkers and isotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks.
Cross-linking combined with mass spectrometry has great potential for determining three-dimensional structures of proteins and protein assemblies. One of the main analytical challenges of this method is the specific detection and identification of the inter-peptide crosslinks in the peptide mixture after enzymatic digestion of the cross-linked protein complex. These inter-peptide crosslinks are important because they provide the critical distance information needed for structural proteomics studies. In this paper, we demonstrate the use of isotopically coded N-terminal modification (ICNTM) in combination with isotopically coded cross-linkers (ICCL) for specific detection of inter-peptide crosslinks. Inter-peptide crosslinks contain two amino termini, compared to one in the case of free peptides, dead-end crosslinks, or intra-peptide crosslinks. Therefore, N-terminal modification with a 1:1 mixture of heavy and light isotopically coded reagents produces inter-peptide crosslinks with a distinct isotopic signature (a 1:2:1 ratio). Modification also occurs at the epsilon-amino groups of non-cross-linked lysine residues, resulting in two modifications per free lysine-containing peptide. However, if ICCL and ICNTM are used together, inter-peptide crosslinks can be distinguished from free lysine-containing peptides. Specialized software has also been developed for the analysis of ICCL + ICNTM experimental data. This procedure, combined with software for data analysis, provides a simple and rapid method for specific detection of inter-peptide crosslinks.
Petrotchenko
Evgeniy V
EV
University of Victoria-Genome British Columbia Protein Centre, Department of Biochemistry and Microbiology, University of Victoria, No. 3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, British Columbia V8Z7X8, Canada.
Serpa
Jason J
JJ
Borchers
Christoph H
CH
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D017434:Protein Structure, Tertiary; D040901:Proteomics; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D003432:Cross-Linking Reagents; D010455:Peptides; D054303:HIV Reverse Transcriptase