Kristine A. Bush

Tandem mass spectrometry (MS/MS) of 28 residue peptides harboring gamma-carboxylated glutamic acid residues, a posttranslational modification of several proenzymes of the blood coagulation cascade, using either collisions or infrared photons results in complete ejection of the gamma-CO2 moieties (-44 Da) before cleavage of peptide-backbone bonds. However, MS/MS using electron capture dissociation (ECD) in a Fourier transform mass spectrometer cleaves backbone bonds without ejecting CO2, allowing direct localization of this labile modification. Sulfated side chains are also retained in ECD backbone fragmentations of a 21-mer peptide, although CAD causes extensive SO3 loss. ECD thus is a unique complement to conventional methods for MS/MS, causing less undesirable loss of side-chain functionalities as well as more desirable backbone cleavages.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and characterization of a new 26.pi.-aromatic thiophene-containing macrocyclic ligandMartin R. Johnson, Douglas C. Miller, Kristine Bush, John J. Becker, and James A. IbersCite this: J. Org. Chem. 1992, 57, 16, 4414–4417Publication Date (Print):July 1, 1992Publication History Published online1 May 2002Published inissue 1 July 1992https://doi.org/10.1021/jo00042a019RIGHTS & PERMISSIONSArticle Views458Altmetric-Citations62LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (566 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.