Limitations of fasting indices in the measurement of insulin sensitivity in Afro-Caribbean adults

In young Afro-Caribbean adults, HOMA-IR compared poorly with other measures of insulin sensitivity. It remains important to determine whether similar findings occur in a more insulin resistant population. However, HOMA-IR correlated with clinical measures of insulin sensitivity (i.e. adiposity), so it may still be useful in epidemiological studies

Identification of Lysine 37 of Histone H2B as a Novel Site of Methylation

Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function

Primary Sequence Confirmation of a Protein Therapeutic Using Top Down MS/MS and MS³

Mass spectrometry has gained widespread acceptance for the characterization of protein therapeutics as a part of the regulatory approval process. Improvements in mass spectrometer sensitivity, resolution, and mass accuracy have enabled more detailed and confident analysis of larger biomolecules for confirming amino acid sequences, assessing sequence variants, and characterizing post translational modifications. This work demonstrates the suitability of a combined approach using intact MS and multistage top down MS/MS analyses for the characterization of a protein therapeutic drug. The protein therapeutic granulocyte-colony stimulating factor was analyzed using a Thermo Fusion Tribrid mass spectrometer using a multistage top down MS approach. Intact mass analysis identified the presence of two disulfide bonds based on exact mass shifts while a combined collision induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) MS/MS approach obtained 80% protein sequence coverage. Isolating MS/MS fragments for MS³ analysis using HCD or CID increased the sequence coverage to 89%. 95% sequence coverage was obtained by reducing human granulocyte-colony stimulating factor (G-CSF) prior to MS/MS and MS³ analysis to specifically target the residues between the disulfide bonds. The use of this combined intact MS and multistage top down MS approach allows for rapid and accurate determination of the primary sequence of a protein therapeutic drug product

Direct Approach for Qualitative and Quantitative Characterization of Glycoproteins Using Tandem Mass Tags and an LTQ Orbitrap XL Electron Transfer Dissociation Hybrid Mass Spectrometer

The application of multiplexed isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL ETD (electron transfer dissociation) hybrid mass spectrometer as a direct approach for qualitative and quantitative characterization of glycoproteins is reported. Bovine fetuin was used as a model glycoprotein in this study. For online liquid chromatography–mass spectrometry (LC–MS) analysis, high-resolution, mass accurate full scan MS spectra were acquired in the Orbitrap mass analyzer followed by data-dependent tandem mass spectrometry (MS/MS) with alternating collision-induced dissociation (CID), ETD, and higher-energy collisional dissociation (HCD) scans. An additional in-source dissociation scan was used as a highly sensitive and selective detection method for eluting glycosylated peptides. By alternatively using three different dissociation methods, 23 glycoforms from all 5 corresponding glycopeptides were identified from a trypsin digest of bovine fetuin. With ETD, labile glycans were retained without any signs of carbohydrate cleavage with concurrent fragmentation of the peptide backbone. Glycosylation sites were clearly localized from the ETD fragmentation data. Glycan structure elucidation was accomplished using CID. The CID experiments generated fragment ions predominantly from cleavage of glycosidic bonds without breaking the peptide bond. Novel to this method, the TMT labeling protocol was modified and adapted for higher labeling efficiency, and a TriVersa NanoMate was used to reinfuse samples to improve ETD and HCD spectra of glycopeptides. Quantification with TMT was verified based on the HCD spectra from multiple nonglycopeptides and glycopeptides. This method can be used as a qualitative and quantitative technique for direct characterization of glycoproteins and has applicability for detection of counterfeit glycoprotein drug products

Characterization of the Aminocarboxycyclopropane-Forming Enzyme CmaC

The biosynthesis of the coronamic acid fragment of the pseudomonal phytotoxin coronatine involves construction of the cyclopropane ring from a γ-chloro-l-allo-Ile intermediate while covalently tethered as a phosphopantetheinyl thioester to the carrier protein CmaD. The cyclopropane-forming catalyst is CmaC, catalyzing an intramolecular displacement of the γ-Cl group by the α carbon. CmaC can be isolated as a Zn2+ protein with about 10-fold higher activity over the apo form. CmaC will not cyclize free γ-chloro amino acids or their S-N-acetylcysteamine (NAC) thioester derivatives but will recognize some other carrier protein scaffolds. Turnover numbers of 5 min-1 are observed for Zn−CmaC, acting on γ-chloro-l-aminobutyryl-S-CmaD, generating 1-aminocyclopropane-1-carbonyl (ACC)-S-CmaD. Products were detected either while still tethered to the phosphopantetheinyl prosthetic arm by mass spectrometry or after thioesterase-mediated release and derivatization of the free amino acid. In D2O, CmaC catalyzed exchange of one deuterium into the aminobutyryl moiety of the γ-Cl-aminoacyl-S-CmaD, whereas the product ACC-S-CmaD lacked the deuterium, consistent with a competition for a γ-Cl-aminobutyryl α-carbanion between reprotonation and cyclization. CmaC-mediated cyclization yielded solely ACC, resulting from C−C bond formation and no azetidine carboxylate from an alternate N−C cyclization. CmaC could cyclize γ,γ-dichloroaminobutyryl to the Cl-ACC product but did not cyclize δ- or ε-chloroaminoacyl-S-CmaD substrates