Development of a nanoLC LTQ Orbitrap Mass Spectrometric Method for Profiling Glycans Derived from Plasma from Healthy, Benign Tumor Control, and Epithelial Ovarian Cancer Patients

Abstract

We report the development of split-less nano-flow liquid chromatography mass spectrometric analysis of glycans chemically cleaved from glycoproteins in plasma. Porous graphitized carbon operating under reverse-phase conditions and an amide-based stationary phase operating under hydrophilic interaction conditions are quantitatively compared for glycan separation. Both stationary phases demonstrated similar column efficiencies and excellent retention time reproducibility without an internal standard to correct for retention time shift. The 95% confidence intervals of the mean retention times were ±4 seconds across 5 days of analysis for both stationary phases; however, the amide stationary phase was observed to be more robust. The high mass measurement accuracy of less than 2 ppm and fragmentation spectra provided highly confident identifications along with structural information. In addition, data are compared amongst samples derived from 10 healthy controls, 10 controls with a differential diagnosis of benign gynecologic tumors, and 10 diseased epithelial ovarian cancer patients (EOC). Two fucosylated glycans were found to be up-regulated in healthy controls and provided an accurate diagnostic value with an area under the receiver operator characteristic curve of 0.87. However, these same glycans provided a significantly less diagnostic value when used to differentiate EOC from benign tumor control samples with an area under the curve of 0.73.

Solvent effects on the retention of oligosaccharides in porous graphitic carbon liquid chromatography

“…Typically, MeCN is used as organic modifier in the running buffer for PGC chromatography of free oligosaccharides [4,5,[18][19][20]. Other solvents are only rarely applied [8,21].…”

Glycoscience aids in biomarker discovery

“…Since these structures are produced by different glycosyltransferases, they often have diverging biological implications (43,44). To address this issue, several recent studies have focused on structure-specific chromatographic profiling of glycans (17,18,21,23,24,45,46). These studies utilize structure-sensitive chromatography (typically, either PGC or HILIC) to separate glycans online prior to MS analysis.…”

“…Once separated, specific glycan structures may be identified based on a retention time library (21,47) or by further fragmentation, e.g. by tandem MS (24). The incorporation of chromatographically-derived structure specificity into existing strategies for MS-based glycan biomarker discovery promises to provide highly sensitive and specific biomarkers for many diseases.…”

“…In relation to glycomic analysis, LC is most commonly used to separate and differentiate between the many potential structural isomers possible in glycan biosynthesis. Isomeric glycoforms of identical mass exhibit differential retention times on structure-sensitive porous graphitized carbon (PGC) (17)(18)(19)(20)(21)(22)(23) or hydrophilic interaction (HILIC) (24,25) stationary phases and may thus be separated orthogonally to MS analysis. Once separated, fragmentation techniques such as tandem MS (MS/MS or MS n ) and/or exoglycosidase digestion may be employed to differentiate structural isomers by monosaccharide structure, connectivity, and linkage (19)(20)(21)(22)(23)(24).…”

A MALDI-MS-based quantitative targeted glycomics (MALDI-QTaG) for total N-glycan analysis

“…For the untargeted glycomics to identify new glycan biomarkers for a specific disease (Bereman et al 2009) or validate follow-on biologics (Wiegandt and Meyer 2014), LC-MS/MS system has been extensively used. However, these analytical instruments are inappropriate for processing hundreds of samples in parallel at routine diagnostic laboratories or industrial settings after biomarkers or overall glycan structures profiling are identified by LC-MS/ MS (Wu et al 2011).…”