Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS.
Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.
Department of Urology and The Proteomics Center, Boston Children's Hospital and Harvard Medical School , Boston, Massachusetts 02115, United States.
Hui
H
Zhou
Peter G
PG
Warren
John W
JW
Froehlich
Richard S
RS
Lee
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D060748:Fetuins / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000652:urine*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D060748:Fetuins; D007074:Immunoglobulin G; D011134:Polysaccharides; D019158:N-Acetylneuraminic Acid