A spin column-free approach to sodium hydroxide-based glycan permethylation

Abstract

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.

Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions*[S]

“…Following development of the ΔS-Cys-Alb assay, an unplanned event occurred in our laboratory in which it was needed. In short, a set of serum samples from stage I lung cancer patients and corresponding age, gender and smoking-status matched controls were undergoing glycan “node” analysis (36–40) as part of an unrelated project. The samples were collected under NIH-sponsorship by seasoned investigators with well-defined standard operating procedures and, on paper, there should not have been any specimen integrity problems.…”

Mass spectrometry for protein sialoglycosylation

“…The further improved solid‐phase permethylation protocol has been applied for high‐throughput quantitative analysis of N ‐glycans . In addition, a microcolumn‐free protocol has been reported very recently …”

“…230,231 In addition, a microcolumnfree protocol has been reported very recently. 232 Although the permethylation for oligosaccharides is quick and effective, unsatisfactory results are often obtained. For example, if permethylation for reduced glycans is performed using DMSO as the ZHANG ET AL.…”

Glycan Node Analysis of Plasma-Derived Extracellular Vesicles

“…This method can be applied directly to complex biological matrices, covers all major classes of glycans, and condenses and captures unique glycan features, such as core fucosylation, α2-6-sialylation, bisecting N-acetylglucosamine (GlcNAc), and β1-6 branching, as single analytical signals, some of which serve as direct molecular surrogates for the activity of specific glycosyltransferases. Therefore, GNA is often capable of providing unique information that may remain undetected with conventional methods [ 30 , 31 , 32 , 37 , 38 , 39 ], and does so on a throughput scale, using instrumentation compatible with the needs of clinical research: a single analyst can reasonably prepare 60–75 samples a week for analysis [ 37 ]; and a single GC-MS instrument can easily analyze hundreds of samples a week.…”