Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification

Abstract

Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.

Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry

“…Plasma contains many more structures than just the seven contained in the standard mixture [8, 43]. Some of these additional glycans are just isomers with, for example , different sialic linkages or branching (Fig.…”

“…2. Much of the diversity of the triantennary glycans shown in this example arises from different sialic acid linkages, which could also be determined via differential derivatization and MALDI-TOF MS [8]. The branching pattern of erythropoietin glycans and many other isomer details can, however, be revealed only by LC–MS.…”

“…Transferrin, human serum albumin (HSA), and human plasma (obtained from buffy coat preparations purchased from the University Clinic for Blood Group Serology and Transfusion Medicine, Graz, Austria) were digested with PNGase F as described in [8]. In short, the sample was denatured in 2% sodium dodecyl sulfate at 60 °C for 10 min.…”

Inflammatory glycoproteins in cardiometabolic disorders, autoimmune diseases and cancer

“…However, quantification by MS is not always reliable and for some samples there can be overlap from isobaric glycans (discrete isomeric glycan structures that possess the same mass) [33]. MS can be used alone or coupled to separation methods such as HPLC, UPLC, HILIC or capillary electrophoresis to increase the sensitivity [35][36][37]. Furthermore, matrix assisted laser desorption-ionization-MS and electrospray ionization-MS are often applied.…”

Prospective applications of ultrahigh resolution proteomics in clinical mass spectrometry

“…One involves high-throughput MALDI-FTICR profiling of biomolecules in clinical cohorts aiming for case-control classifications. Species of interest are serum peptides and -proteins up to 15 kDa that are obtained after single-step sample cleanup procedures [60,61], released N-glycans that are derived and purified from serum proteins [62], or complex glycopeptide mixtures that are obtained from various immunoglobulins [63]. In addition, we have applied an MALDI-FTICR profiling method that builds on the standard MALDI-TOF approach (Biotyper ™ ) for typing and identifying bacteria and yeasts as described in the previous section.…”