Unexpected products from the reaction of the synthetic cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate), DTSSP with peptides.
Synthetic cross-linking reagents, such as 3,3'-dithiobis(sulfosuccinimidyl propionate), DTSSP, can react with sidechains of amino acids that are within close proximity. Identification of cross-linked residues provides insight into the folded structures of proteins. However, analysis of proteolytic digests of proteins cross-linked with commercially available DTSSP is difficult because many ions cannot be attributed to reported reactions of DTSSP. To better understand the reactivity of DTSSP, products from the reaction of DTSSP with several model peptides were analyzed by HPLC electrospray ionization mass spectrometry (ESIMS). Several products not previously reported were identified. Sources for these unexpected products were traced to reaction of DTSSP with contaminant ammonium ions in the buffer, to reaction of contaminants present in the commercial DTSSP reagent, and to reactivity of DTSSP with serine and tyrosine residues. In addition, the collision-induced-dissociation (CID) of peptides modified by DTSSP was investigated. These results showed that certain DTSSP-peptide adducts easily undergo in-source fragmentation to give additional unexpected ions. This study of the reactions of DTSSP with model peptides has revealed the major types of ions that are likely to be found in proteolytic digests of proteins cross-linked with DTSSP, thereby facilitating identification of the cross-linked residues that can provide information about the three-dimensional structures of folded proteins.
Swaim
Catherine L
CL
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
Smith
Jean B
JB
Smith
David L
DL
D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013388:Succinimides / Q000737:chemistry*
D000588:Amines; D003432:Cross-Linking Reagents; D010455:Peptides; D000644:Quaternary Ammonium Compounds; D013388:Succinimides; D012694:Serine; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)