Simultaneous quantification of N- and O-glycans using a solid-phase method

Abstract

Glycosylation has a pivotal role in a diverse range of biological activities, modulating the structure and function of proteins. Glycogens coupled to the nitrogen atom (N-linked) of asparagine side chains or to the oxygen atom (O-linked) of serine and threonine side chains represent the two major protein glycosylation forms. N-glycans can be released by glycosidases, whereas O-glycans are often cleaved by chemical reaction. However, it is challenging to combine these enzymatic and chemical reactions in order to analyze both N- and O-glycans. We recently developed a glycoprotei n immobilization for glycan extraction (GIG) method that allows for the simultaneous analysis of N- and O-glycans on a solid support. GIG enables quantitative analysis of N-glycans and O-glycans from a single specimen and can be applied to a high-throughput automated platform. Here we provide a step-by-step GIG protocol that includes procedures for (i) protein immobilization on an aldehyde-active solid support by reductive amination; (ii) stabilization of fragile sialic acids by carbodiimide coupling; (iii) release of N-glycans by PNGase F digestion; (iv) release of O-glycans by β-elimination using ammonia in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP) to prevent alditol peeling from O-glycans; (v) mass spectrometry (MS) analysis; and (vi) data analysis for identification of glycans using in-house developed software (GIG Tool; free to download via http://www.biomarkercenter.org/gigtool). The GIG tool extracts precursor masses, oxonium ions and glycan fragments from tandem (liquid chromatography (LC)–MS/MS) mass spectra for glycan identification, and reporter ions from quaternary amine containing isobaric tag for glycan (QUANTITY) isobaric tags are used for quantification of the relative abundance of N-glycans. The GIG protocol takes ~3 d.

Sensitive and robust MALDI-TOF-MS glycomics analysis enabled by Girard's reagent T on-target derivatization (GTOD) of reducing glycans

“…4D) GSLs-glycans, 37, 19, 13 and 10 different compositions were identified, whereas only 29, 11, 7 and 9 compositions were identified in the MS profiles without GTOD treatment. The compositions assigned based on MS data are listed in Tables S4–8, which are consistent with previous reports [14,52,59,60]. The compositions identified only with GTOD (labeled with blue characters) were all assigned to low-abundance glycans.…”

“…Glycomics is the systematic study of glycan structures, including monosaccharide compositions, glycan sequence, branching pattern and linkage specifications, which are essential to the functions of glycoconjugates [11–14]. Nevertheless, glycomics analysis is inherently challenging due to their structure complexity and heterogeneity, low natural abundance and the lack of amplification methods [5,14,15]. …”

“…Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become widely used for high throughput profiling of glycans with high heterogeneity and low abundance [14,16–21]. MALDI-TOF-MS generates dominantly singly charged ions and detects a large mass range.…”

“…MALDI-TOF-MS generates dominantly singly charged ions and detects a large mass range. Sample preparation is simple and high throughput analysis can be carried out easily and quickly [14,22,23]. However, MALDI-MS analysis of underivatized glycans is limited by their low ionization efficiency.…”

Mass spectrometry for protein sialoglycosylation

“…The two‐step derivatization results in a mass increase of 13 Da for each sialic acid in comparison to only permethylated glycans. Another amidation protocol, based on p ‐toluidine and EDC, can stabilize sialic acids at the protein level and provide a significantly increased MS signal for sialylated O ‐glycans …”