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Identification of Cross-linked Peptides Using Isotopomeric Cross-linkers.
- Source :
-
Journal of the American Society for Mass Spectrometry [J Am Soc Mass Spectrom] 2019 Sep; Vol. 30 (9), pp. 1643-1653. Date of Electronic Publication: 2019 Jun 05. - Publication Year :
- 2019
-
Abstract
- Chemical cross-linking combined with mass spectrometry (CL-MS) is a powerful method for characterizing the architecture of protein assemblies and for mapping protein-protein interactions. Despite its proven utility, confident identification of cross-linked peptides remains a formidable challenge, especially when the peptides are derived from complex mixtures. MS cleavable cross-linkers are gaining importance for CL-MS as they permit reliable identification of cross-linked peptides by whole proteome database searching using MS/MS information. Here we introduce a novel class of MS cleavable cross-linkers called isotopomeric cross-linkers (ICLs), which allow for confident and efficient identification of cross-linked peptides by whole proteome database searching. ICLs are simple, symmetrical molecules that asymmetrically incorporate heavy and light stable isotopes into the two arms of the cross-linker. As a result of this property, ICLs automatically generate pairs of isotopomeric cross-linked peptides, which differ only by the positions of the heavy and light isotopes. Upon fragmentation during MS analysis, these isotopomeric cross-linked peptides generate unique isotopic doublet ions that correspond to the individual peptides in the cross-link. The doublet ion information is used to determine the masses of the two cross-linked peptides from the same MS2 spectrum that is also used for peptide spectrum matching (PSM) by sequence database searching. Here we present the rationale for and mechanism of cross-linked peptide identification by ICL-MS. We describe the synthesis of the ICL-1 reagent, the ICL-MS workflow, and the performance characteristics of ICL-MS for identifying cross-linked peptides derived from increasingly complex mixtures by whole proteome database searching.
- Subjects :
- Cross-Linking Reagents chemical synthesis
Isotopes chemistry
Proteome analysis
Proteome chemistry
RNA Polymerase II analysis
RNA Polymerase II chemistry
Serum Albumin, Bovine analysis
Serum Albumin, Bovine chemistry
Spectrometry, Mass, Electrospray Ionization methods
Cross-Linking Reagents chemistry
Mass Spectrometry methods
Peptides analysis
Peptides chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1879-1123
- Volume :
- 30
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Journal of the American Society for Mass Spectrometry
- Publication Type :
- Academic Journal
- Accession number :
- 31168746
- Full Text :
- https://doi.org/10.1007/s13361-019-02253-z