Mass Spectrometric Identification of a Novel Factor XIIIa Cross-Linking Site in Fibrinogen.

Mariya E Semkova,J Justin Hsuan

doi:10.3390/proteomes9040043

Abstract

Transglutaminases are a class of enzymes that catalyze the formation of a protein:protein cross-link between a lysine and a glutamine residue. These cross-links play important roles in diverse biological processes. Analysis of cross-linking sites in target proteins is required to elucidate their molecular action on target protein function and the molecular specificity of different transglutaminase isozymes. Mass-spectrometry using settings designed for linear peptide analysis and software designed for the analysis of disulfide bridges and chemical cross-links have previously been employed to identify transglutaminase cross-linking sites in proteins. As no control peptide with which to assess and improve the mass spectrometric analysis of TG cross-linked proteins was available, we developed a method for the enzymatic synthesis of a well-defined transglutaminase cross-linked peptide pair that mimics a predicted tryptic digestion product of collagen I. We then used this model peptide to determine optimal score thresholds for correct peptide identification from y- and b-ion series of fragments produced by collision-induced dissociation. We employed these settings in an analysis of fibrinogen cross-linked by the transglutaminase Factor XIIIa. This approach resulted in identification of a novel cross-linked peptide in the gamma subunit. We discuss the difference in behavior of ions derived from different cross-linked peptide sequences and the consequent demand for a more tailored mass spectrometry approach for cross-linked peptide identification compared to that routinely used for linear peptide analysis.

Highlights

The first aim of this of the study was to synthesize a cross-linked peptide pair with known sequences that would allow (i) an analysis of the collision-induced fragmentation behavior of a model TG cross-linked peptide, and (ii) thresholding of software output scores for different putative sites identified via analysis of fragmentation data
TG cross-linked peptides have previously been identified in a tryptic digest of transglutaminase 2 (TG2) cross-linked lacritin using the MassMatrix software [31]; the presence of multiple unassigned peaks with relatively high intensities in the fragmentation spectra reduced confidence, and the threshold and sample scores used for positive identification were not reported
Our hypothesis that a thorough investigation of the fragmentation behavior of TG cross-linked peptides is necessary for their reliable identification was confirmed

Summary

Introduction

Formation of a protein polymer (de novo direct polymerization) [6], stabilization of non-covalent assemblies by introducing covalent bonds between adjacent subunits—known as enzymatic “spot welding”—or conformational restriction of a folded polypeptide takes place [1,7,8,9]. Apart from their cross-linking activity, this class of enzymes has been reported to carry out other enzymatic activities including protein phosphorylation [10], glutamine deamidation [11], amine incorporation [12] and esterification [13].

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Conclusion