Annotation of a Serum N-Glycan Library for Rapid Identification of Structures

Abstract

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible due to the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC/MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples.

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.…”

“…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).…”

“…Due to the structurally complex nature of glycosylation and the functional impact of small structural variations in glycans, much importance is placed on differentiating isomeric glycoforms (6,17,43,86). In particular, porous graphitized carbon (PGC), a mixed-mode stationary phase popularly used in carbohydrate analysis, has been applied extensively to separation of glycan isomers (17,18,21,45,46). Lately, PGC nano-LC has been extended towards the separation of glycopeptides (22,59,87).…”

“…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).…”

Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography–electrospray ionization-mass spectrometry with graphitic carbon stationary phase

“…HILIC columns, either with amide or zwitterionic functionalities, as well as PGC stationary phases have been extensively used for the separation of glycans released from proteins [5,[10][11][12][13][14][15][16][17]. Structureretention time reference libraries have been established for both HILIC and PGC glycan separations to simplify identification of isobaric glycans [18,19]. The successful use of PGC as stationary phase for glycan separation was reported for neat glycans [20,21], neat glycans reduced to the alditols [13,22], and labeled glycans.…”