Naturally occurring asymmetric and triantennay N -glycans can be isolated from hen egg yolk glycopeptides.

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N-glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N-glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N-glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N-glycans as an additional or orthogonal method to current analytical approaches.

Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients' urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA. SIGNIFICANCE: The detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and non-cleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies.

A newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum N-glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum N-glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively. This strategy allowed efficient separation of specific positional isomers on reversed-phase liquid chromatography–fluorescence detection–mass spectrometry (RPLC-FD-MS) and complemented the previous glycomics data based on matrix-assisted laser desorption/ionization (MALDI)-MS that did not include such separations. The results from comparing pre-operative CRC to post-operative samples were in agreement with studies that identified a decrease in di-antennary structures with core fucosylation and an increase in sialylated tri- and tetra-antennary N-glycans in CRC patient sera. Pre-operative abundances of N-glycans showed good performance for the classification of adenocarcinoma and led to the revisit of the previous MALDI-MS dataset with regard to histological and clinical data. This strategy has the potential to monitor patient profiles before, during, and after clinical events such as treatment, therapy, or surgery and should also be further explored.

Isolates of the E. coli O101 serogroup collected from bacteremia patients were demonstrated to harbor different glycoforms of the O-antigen polysaccharide. Investigation by high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy of the O-antigen structure of the reference E. coli O101 strain H510a and of one naturally occurring variant (Onovel32) confirmed the previously reported simultaneous presence of O-polysaccharide repeat units made up of disaccharides PS1 and PS2. A newly discovered O101 glycoform was shown to be composed of the same backbones modified with a previously unrecognized structure having an additional terminal 4-O-methyl N-acetyl-galactosamine (4-O-Me-GalNAc) residue on the non-reducing end of the PS2 backbone. Another naturally occurring variant (Onovel32MT-) does not contain any PS2 repeat units, nor any 4-O-Me-GalNAc capping. Analyses indicated that the polysaccharide glycoforms from these three O101 strains varied in terms of their ratios of PS1 to PS2 repeat units and the amount of 4-O-Me-GalNAc terminal residue. From the Smith degradation data of the reference O101 strain and the Onovel32MT- variant, it is hypothesized that the PS1 and PS2 repeat units join into a PS1-PS2 block copolymer, which is formed by the addition of the PS2 backbone on the already existing PS1 core backbone. Finally, 4-O-Me-GalNAc acts as a terminal capping residue on the PS2 backbone. Such block copolymer structures have hitherto been reported in galactans of Klebsiella and in Brucella. To our knowledge, this is the first reported occurrence of such a block copolymer O-antigen in E. coli.