Deglycosylation and Sample Cleanup Method for Mass Spectrometry Analysis of N-linked Glycans

l ycosylation is one of the most important types of posttranslational modification (PTM) in proteins. Due to the high d e g ree of hetero g e n e i t y, the characterization of glycans is a challenging task. Mass spectro m e t ry (MS) is a primary tool for biopolymer analysis; howe ve r, the characterization of (native) glycans is complicated by the time-consuming sample preparation and their poor MS ionization efficiency. A typical sample preparation method for MS i n vo l ves a chemical or enzymatic cleavage of glycans, followed by salts, surfactants, and protein residues re m oval. Purified native glycans can be directly analyzed by MALDI–TOF MS. The efficient sample deglycosylation is a key re q u i rement for a successful and sensitive glycan analysis. Ne ve rtheless, the quantitative g l ycan release (e.g., using enzymes) is rarely achieved, since the glyc osylated sites of the proteins are often obstructed by the protein seco n d a ry and tert i a ry stru c t u re . The goal of this work was to develop a rapid and efficient deglycosylation of N-linked glyc o p roteins with a glycosidase (PNGase F) aided with the enzyme-friendly surfactant, RapiGestTM SF. This was f o l l owed with a novel micro-scale hyd rophilic-interaction chromatography (HILIC) solid-phase extraction (SPE) plate (Wa t e r s® MassPREPTM HILIC mElution Plate) for a rapid sample cleanup prior to MALDI MS analysis using highly purified MALDI matrix (Wa t e r s® MassPREPTM MALDI Matrix, DHB).