Abstract
Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F (PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis.
Highlights
Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases
Sequencing the human genome and that of various pathogens has opened the door for proteomics, which has dramatically facilitated the search for diagnostic biomarkers
Using an optimized protocol for quantitative glycomics, we revealed for the first time the absolute concentrations of major N-glycans occurring in human serum whole glycoproteins
Summary
1-Butanol, ammonium bicarbonate, and sodium phosphate buffer were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). 2-Hydroxyl-3-sulfopropyl dodecanoate (HSD), 3-methyl-1-p-tolyltriazene (MTT), and sodium cyanoborohydride were purchased from Aldrich. 1-Butanol, ammonium bicarbonate, and sodium phosphate buffer were purchased from Kanto Chemical Co., Inc. The precipitate was resuspended in 0.1 M sodium phosphate buffer (pH 7.0) containing 1 M ammonium sulfate and 1 mM EDTA and centrifuged. Both forms of N␣-((aminooxy)acetyl)tryptophanylarginine methyl ester (aoWR), aoWR(H) and aoWR(D), were prepared as described previously [16]. Human sera from two normal subjects (males, 73 and 69 years old, designated as normal sera A and B, respectively) were purchased from Genomics Collaborative Inc. Human serum was purchased from Sigma-Aldrich and was designated as normal serum C. A serum sample of a rheumatoid arthritis (RA) patient (female, 52 years old) was obtained from the Department of Orthopaedic Surgery, Hokkaido University School of Medicine (Sapporo, Japan) under the permission of the Commission of Bioethics
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