An isotopically coded CID-cleavable biotinylated cross-linker for structural proteomics.
Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of cross-links. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase.
Petrotchenko
Evgeniy V
EV
University of Victoria-Genome British Columbia Proteomics Centre, Department of Biochemistry and Microbiology, University of Victoria, Vancouver Island Technology Park Victoria, British Columbia, Canada.
Serpa
Jason J
JJ
Borchers
Christoph H
CH
D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D019921:Biotinylation; D002845:Chromatography / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology*; D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
D003432:Cross-Linking Reagents; D010455:Peptides; D011506:Proteins; D020543:Proteome; D013388:Succinimides; C559601:cyanurbiotindipropionylsuccinimide; C087949:N-propionylsuccinimide; D001710:Biotin; D054303:HIV Reverse Transcriptase