Identification of respective lysine donor and glutamine acceptor sites involved in factor XIIIa-catalyzed fibrin α chain cross-linking.
Factor XIIIa-catalyzed ε-(γ-glutamyl)-lysyl bonds between glutamine and lysine residues on fibrin α and γ chains stabilize the fibrin clot and protect it from mechanical and proteolytic damage. The cross-linking of γ chains is known to involve the reciprocal linkages between Gln(398) and Lys(406). In α chains, however, the respective lysine and glutamine partners remain largely unknown. Traditional biochemical approaches have only identified the possible lysine donor and glutamine acceptor sites but have failed to define the respective relationships between them. Here, a differential mass spectrometry method was implemented to characterize cross-linked α chain peptides originating from native fibrin. Tryptic digests of fibrin that underwent differential cross-linking conditions were analyzed by high resolution Fourier transform mass spectrometry. Differential intensities associated with monoisotopic masses of cross-linked peptides were selected for further characterization. A fit-for-purpose algorithm was developed to assign cross-linked peptide pairs of fibrin α chains to the monoisotopic masses relying on accurate mass measurement as the primary criterion for identification. Equipped with hypothesized sequences, tandem mass spectrometry was then used to confirm the identities of the cross-linked peptides. In addition to the reciprocal cross-links between Gln(398) and Lys(406) on the γ chains of fibrin (the positive control of the study), nine specific cross-links (Gln(223)-Lys(508), Gln(223)-Lys(539), Gln(237)-Lys(418), Gln(237)-Lys(508), Gln(237)-Lys(539), Gln(237)-Lys(556), Gln(366)-Lys(539), Gln(563)-Lys(539), and Gln(563)-Lys(601)) on the α chains of fibrin were newly identified. These findings provide novel structural details with respect to the α chain cross-linking compared with earlier efforts.
Molecular Biomarkers, Merck Research Laboratories, Rahway, New Jersey 07065, USA. weixun_wang@merck.com
Weixun
W
Wang
D001665:Binding Sites / Q000502:physiology; D026122:Factor XIIIa / Q000737:chemistry* / Q000378:metabolism; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D010455:Peptides; D005973:Glutamine; D005337:Fibrin; D026122:Factor XIIIa; D008239:Lysine