Nonprotein based enrichment method to analyze peptide cross-linking in protein complexes.
Cross-linking analysis of protein complexes and structures by tandem mass spectrometry (MS/MS) has advantages in speed, sensitivity, specificity, and the capability of handling complicated protein assemblies. However, detection and accurate assignment of the cross-linked peptides are often challenging due to their low abundance and complicated fragmentation behavior in collision-induced dissociation (CID). To simplify the MS analysis and improve the signal-to-noise ratio of the cross-linked peptides, we developed a novel peptide enrichment strategy that utilizes a cross-linker with a cryptic thiol group and using beads modified with a photocleavable cross-linker. The functional cross-linkers were designed to react with the primary amino groups in proteins. Human serum albumin was used as a model protein to detect intra- and intermolecular cross-linkages. Use of this protein-free selective retrieval method eliminates the contamination that can result from avidin-biotin based retrieval systems and simplifies data analysis. These features may make the method suitable to investigate protein-protein interactions in biological samples.
Yan
Funing
F
Department of Biochemistry, and Biodefense Proteomics Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA. fyan@aecom.yu.edu
Che
Fa-Yun
FY
Rykunov
Dmitry
D
Nieves
Edward
E
Fiser
Andras
A
Weiss
Louis M
LM
Hogue Angeletti
Ruth
R
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010782:Photolysis; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000379:methods*
D003432:Cross-Linking Reagents; D010455:Peptides; D011506:Proteins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds