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Deamidation and Transamidation of Substance P by Tissue Transglutaminase Revealed by Electron‐Capture Dissociation Fourier Transform Mass Spectrometry
Author(s) -
Fornelli Luca,
Schmid Adrien W.,
Grasso Luigino,
Vogel Horst,
Tsybin Yury O.
Publication year - 2011
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201002483
Subject(s) - deamidation , chemistry , tissue transglutaminase , peptide , mass spectrometry , fourier transform ion cyclotron resonance , tandem mass spectrometry , electron capture dissociation , glutamine , enzyme , amino acid , biochemistry , stereochemistry , chromatography
Tissue transglutaminase (tTGase) catalyzes both deamidation and transamidation of peptides and proteins by using a peptidyl glutamine as primary substrate. A precise consensus sequence for the enzyme is unknown and the ratio between deamidated and transamidated (or cross‐linked) reaction products is highly substrate‐dependent. Due to its overlapping body distribution with tTGase and ease of manipulation with tandem mass spectrometry, we used the neuropeptide substance P as a model to investigate the associated enzymatic kinetics and reaction products. Online liquid‐chromatography Fourier‐transform ion‐cyclotron‐resonance mass spectrometry (FT‐ICR MS) combined with electron‐capture dissociation (ECD) was employed to study the tTGase‐induced modifications of substance P. A particular strength of ECD for peptide‐enzyme reaction product monitoring is its ability to distinguish isomeric amino acids, for example, Glu and iso‐Glu, by signature product ions. Our studies show that the primary reaction observed is deamidation, with the two consecutive glutamine residues converted sequentially into glutamate: first Gln 5 , and subsequently Gln 6 . We then applied ECD FT‐ICR MS to identify the transamidation site on an enzymatically cross‐linked peptide, which turned out to correspond to Gln 5 . Three populations of substance‐P dimers were detected that differed by the number of deamidated Gln residues. The higher reactivity of Gln 5 over Gln 6 was further confirmed by cross‐linking SP with monodansylcadaverine (MDC). Overall, our approach described herein is of a general importance for mapping both enzymatically induced post‐translational protein modifications and cross‐linking. Finally, in vitro Ca‐signaling assays revealed that the main tTGase reaction product, the singly deamidated SP (RPKPEQFFGLM‐NH 2 ), has increased agonist potency towards its natural receptor, thus confirming the biologically relevant role of deamidation.