Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues—yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p < 0.05), but overall yield of O-glycans was similar or slightly diminished (p < 0.05 for tetrasaccharides or smaller). When the SCF procedure was employed upstream to hydrolysis, reduction and acetylation for glycan linkage analysis of pooled glycans from unfractionated blood plasma, analytical reproducibility was on par with that from previous spin column-based “glycan node” analysis results. When applied to blood plasma samples from stage III–IV breast cancer patients (n = 20) and age-matched controls (n = 20), the SCF procedure facilitated identification of three glycan nodes with significantly different distributions between the cases and controls (ROC c-statistics > 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens.

•Albumin glycation increases ex vivo in blood plasma samples stored above −30 °C.•Increased atmospheric oxygen content leads to higher ex vivo albumin glycation.•Increased sample surface area-to-volume leads to higher ex vivo albumin glycation.•Autoxidative processes likely account for increased ex vivo albumin glycation.Ex vivo protein modifications occur within plasma and serum (P/S) samples due to prolonged exposure to the thawed state—which includes temperatures above −30 °C. Herein, the ex vivo glycation of human serum albumin from healthy and diabetic subjects was monitored in P/S samples stored for hours to months at −80 °C, −20 °C, and room temperature, as well as in samples subjected to multiple freeze-thaw cycles, incubated at different surface area-to-volume ratios or under different atmospheric compositions. A simple dilute-and-shoot method utilizing trap-and-elute LC-ESI-MS was employed to determine the relative abundances of the glycated forms of albumin—including forms of albumin bearing more than one glucose molecule. Significant increases in glycated albumin were found to occur within hours at room temperature, and within days at −20 °C. These increases continued over a period of 1–2 weeks at room temperature and over 200 days at −20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.

Dysregulated glycotransferase enzymes in cancer cells produce aberrant glycans—some of which can help facilitate metastases. Within a cell, individual glycotransferases promiscuously help to construct dozens of unique glycan structures, making it difficult to comprehensively track their activity in biospecimens—especially where they are absent or inactive. Here, we describe an approach to deconstruct glycans in whole biospecimens then analytically pool together resulting monosaccharide-and-linkage-specific degradation products (“glycan nodes”) that directly represent the activities of specific glycotransferases. To implement this concept, a reproducible, relative quantitation-based glycan methylation analysis methodology was developed that simultaneously captures information from N-, O-, and lipid linked glycans and is compatible with whole biofluids and homogenized tissues; in total, over 30 different glycan nodes are detectable per gas chromatography–mass spectrometry (GC-MS) run. Numerous nonliver organ cancers are known to induce the production of abnormally glycosylated serum proteins. Thus, following analytical validation, in blood plasma, the technique was applied to a group of 59 lung cancer patient plasma samples and age/gender/smoking-status-matched non-neoplastic controls from the Lung Cancer in Central and Eastern Europe (CEE) study to gauge the clinical utility of the approach toward the detection of lung cancer. Ten smoking-independent glycan node ratios were found that detect lung cancer with individual receiver operating characteristic (ROC) c-statistics ranging from 0.76 to 0.88. Two glycan nodes provided novel evidence for altered ST6Gal-I and GnT-IV glycotransferase activities in lung cancer patients. In summary, a conceptually novel approach to the analysis of glycans in unfractionated human biospecimens has been developed that, upon clinical validation for specific applications, may provide diagnostic and/or predictive information in glycan-altering diseases.

As a posttranslational protein modification, cysteine sulfenic acid (Cys-SOH) is well established as an oxidative stress-induced mediator of enzyme function and redox signaling. Data presented herein show that protein Cys-SOH forms spontaneously in air-exposed aqueous solutions of unfolded (disulfide-reduced) protein in the absence of added oxidizing reagents, mediating the oxidative disulfide bond formation process key to in vitro, nonenzymatic protein folding. Molecular oxygen (O2) and trace metals [e.g., copper(II)] are shown to be important reagents in the oxidative refolding process. Cys-SOH is also shown to play a role in spontaneous disulfide-based dimerization of peptide molecules containing free cysteine residues. In total, the data presented expose a chemically ubiquitous role for Cys-SOH in solutions of free cysteine-containing protein exposed to air.

Mass spectrometric evidence presented here characterizes the genotype-dependent glycosylation patterns for each of the three major allele products of Vitamin D Binding Protein found in the general human population. Findings based on the analysis of over 100 individual plasma samples demonstrated that all DBP allele products, except GC*2, are modified (10−25 mol%) with a linear (NeuNAc)1(Gal)1(GalNAc)1 trisaccharide and, to a much lesser extent (1−5 mol%) with a trisaccharide-independent (Gal)1(GalNAc)1 dissaccharide. GC*2 protein contains the disaccharide but remains completely free of the trisaccharide, even in heterozygous individuals possessing a second gene product that is modified with the trisaccharide. Thus, all allelic forms of DBP except GC*2 possess two independent O-glycosylation sites occupied by separate, yet consistently isomass oligosaccharides and, despite a consensus sequence, lack N-glycosylation.