Characterization of the human platelet N- and O-glycome upon storage using tandem mass spectrometry.

Changes in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N- and O-linked glycans in freshly isolated and 7-day room temperature-stored platelet concentrates.

Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671.

Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation.

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined.

Platelets support extracellular sialylation by supplying the sugar donor substrate.

Sizable pools of freely circulating glycosyltransferases are in blood, but understanding their physiologic contributions has been hampered because functional sources of sugar donor substrates needed to drive extracellular glycosylation have not been identified. The blood-borne ST6Gal-1 produced and secreted by the liver is the most noted among the circulatory glycosyltransferases, and decorates marrow hematopoietic progenitor cells with α2,6-linked sialic acids and restricts blood cell production. Platelets, upon activation, secrete a plethora of bioactive molecules including pro- and anti-inflammatory mediators.

Structural documentation of glycan epitopes: sequential mass spectrometry and spectral matching.

Documenting mass spectral data is a fundamental aspect of accepted protocols. In this report, we contrast MS(n) sequential disassembly spectra obtained from natural and synthetic glycan epitopes. The epitopes considered are clusters found on conjugate termini of lipids and N- and O-glycans of proteins.

Profiling and characterization of sialylated N-glycans by 2D-HPLC (HIAX/PGC) with online orbitrap MS/MS and offline MSn.

Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor-Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins.

Anomalous N-glycan structures with an internal fucose branched to GlcA and GlcN residues isolated from a mollusk shell-forming fluid.

This report describes the structural details of a unique N-linked valence epitope on the major protein within the extrapallial (EP) fluid of the mollusk, Mytilus edulis. Fluids from this area are considered to be responsible for shell expansion by a self-assembly process that provides an organic framework for the growth of CaCO3 crystals. Previous reports from our laboratories have described the purification and amino acid sequence of this EP protein, which was found to be a glycoprotein (EPG) of approximately 28 KDa with 14.

Toward a platform for comprehensive glycan sequencing.

From a series of recently published reports, an analytical platform has been proposed for a quantitative and qualitative measure of N- and O-glycosylation, complete with peptide-glycan connectivity and detailed structural understanding. As distant as this may appear, a best methods approach will appear that must move us beyond the cartoon stage of structural understanding. Thus, with this unifying goal in mind, we summarize a series of individually promising first phase protocols of sample preparation (release, purification, and quantification) that remain congruent with a concluding phase (methylation and MS(n)) for documented structural detail.

Isomer and glycomer complexities of core GlcNAcs in Caenorhabditis elegans.

Analysis of protein glycosylation within the nematode Caenorhabditis elegans has revealed an abundant and unreported set of core chitobiose modifications (CCMs) to N-linked glycans. With hydrazine release, an array of glycomers and isobars were detected with hexose extensions on the 3- and 3,6-positions of the penultimate and reducing terminus, respectively. A full complement of structures includes a range of glycomers possessing a Galbeta(1-4)Fuc disaccharide at the 3- and 6-positions of the protein-linked GlcNAc.

Caenorhabditis elegans triple null mutant lacking UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

We have previously reported, from the nematode worm Caenor-habditis elegans, three genes (gly-12, gly-13 and gly-14) encoding enzymically active UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid, paucimannose and complex N-glycan synthesis. We now describe a worm with null mutations in all three GnT I genes, gly-14 (III);gly-12 gly-13 (X) (III and X refer to the chromosome number). The triple-knock-out (TKO) worms have a normal phenotype, although they do not express GnT I activity and do not synthesize 31 paucimannose, complex and fucosylated oligomannose N-glycans present in the wild-type worm.