The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed. Molecular & Cellular Proteomics

Aberrant glycosylation of proteins and lipids has been implicated in many human diseases, thus prompting the need for reliable analytical methods that permit reliable quantification of glycans originating from biological specimens. MS of permethylated glycans is currently employed to monitor disease related aberrant glycosylation of proteins and lipids. However, enhancing the sensitivity of this type of analysis is still needed. Here, analysis of permethylated glycans at enhanced sensitivity is attained through miniaturized solid-phase permethylation as well as on-line solid-phase purification. Solid-phase permethylation method was miniaturized by reducing the amount of sodium hydroxide beads (one-third the original amount) packed in microspin columns. The efficiency of glycan permethylation was not adversely affected by this reduction. On-line solid-phase purification of permethylated N-glycans derived from model glycoproteins, such as fetuin, α-1 acid glycoprotein and ribonuclease B, offered more sensitive and reproducible results than off-line liquid-liquid and solid-phase extractions. On-line solid-phase purification method described here permitted a seventy-five percent increase in signal intensities of permethylated glycans relative to off-line purification methods. This is mainly due to the minimized sample handling associated with an on-line cleaning procedure. The efficiency and utility of on-line solid-phase purification was also demonstrated for N-glycans derived from human blood serum. On-line solid-phase purification permitted the detection of 66 N-glycan structures, while only 58 glycan structures were detected in the case of samples purified through liquid-liquid extraction. The intensities of the 58 structures that were detected in both cases were seventy-five percent higher for samples that were purified through the described method.

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan structures was determined to be 30% while it was found to be 35% for either fucosylated or sialylated structures The optimum CE for mannose and complex type N-glycan structures was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan structures in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these structures was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitudes. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan structures enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Summary For decades, the association between aberrant glycosylation and many types of cancers has been shown. However, defining the changes of glycan structures has not been demonstrated until recently. This has been facilitated by the major advances in mass spectrometry and separation science which allowed the detailed characterization of glycan changes associated with cancer. Mass spectrometry glycomics methods have been successfully employed to compare the glycomic profiles of different human specimen collected from disease-free individuals and patients with cancer. Additionally, comparing the glycomic profiles of glycoproteins purified from specimen collected from disease-free individuals and patients with cancer has also been performed. These types of glycan analyses employing mass spectrometry or liquid-chromatography mass spectrometry allowed the characterization of native, labeled, and permethylated glycans. This review discusses the different glycomic and glycoproteomic methods employed for defining glycans as cancer biomarkers of different organs, including breast, colon, esophagus, liver, lung, ovarian, pancreas and prostate.

RATIONALE Mass spectrometry based comparative glycomics is essential for disease biomarker discovery. However, developing a reliable quantification method is still a challenging task. METHODS We here report an isotopic labeling strategy employing stable isotopic iodomethane for comparative glycomic profiling by LC-ESI-MS. N-Glycans released from model glycoproteins and blood serum samples were permethylated with iodomethane (“light”) and iodomethane-d1 or -d3 (“heavy”) reagents. Permethylated samples were then mixed at equal volumes prior to LC-ESI-MS analysis. RESULTS Peak intensity ratios of N-glycans isotopically permethylated (Heavy/Light, H/L) were almost equal to the theoretical values. Observed differences were mainly related to the purity of “heavy” iodomethane reagents (iodomethane-d1 or -d3). The data suggested the efficacy of this strategy to simultaneously quantify N-glycans derived from biological samples representing different cohorts. Accordingly, this strategy is effective in comparing multiple samples in a single LC-ESI-MS analysis. The potential of this strategy for defining glycomic differences in blood serum samples representing different esophageal diseases was explored. CONCLUSIONS LC-ESI-MS comparative glycomic profiling of isotopically permethylated N-glycans derived from biological samples and glycoproteins reliably defined glycan changes associated with biological conditions or glycoproteins expression. As a biological application, this strategy permitted the reliable quantification of glycomic changes associated with different esophageal diseases, including high grade dysplasia, Barrett’s disease and esophageal adenocarcinoma.