Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein

• Published in: Journal of mass spectrometry. Vol. 35; no. 12; pp. 1416 - 1424
• Main Authors: Faull, Kym F., Johnson, Jeffrey, Kim, Moon Jae, To, Trang, Whitelegge, Julian P., Stevens, Richard L., Fluharty, Claire B., Fluharty, Arvan L.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.12.2000; Wiley
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Asparagine - chemistry; Biological and medical sciences; Carbohydrate Conformation; cerebroside sulfate activator protein; Chromatography, High Pressure Liquid; electrospray mass spectrometry; exoglycosidase digestion; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycoproteins - chemistry; Glycoproteins - urine; Humans; Kidney - chemistry; matrix-assisted laser desorption/ionization; Molecular Structure; oligosaccharide structure; Proteins; saposin B; Saposins; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipid Activator Proteins; Swine; trimethyl-p-aminophenyl derivatization; Molecular form; HPLC chromatography; Matrix assisted laser desorption ionization; Derivatization; Electrospray; Kidney; Sample preparation; Ungulata; Human; Urine; Enzyme; Oligosaccharide; Glycoprotein; Animal origin; Protein; Pig; Vertebrata; Mammalia; Analysis method; Glycosidases; Mass spectrometry MS/MS; Hydrolases; Artiodactyla; O-Glycosidases; Structural analysis
• ISSN: 1076-5174; 1096-9888
• DOI: 10.1002/1096-9888(200012)35:12<1416::AID-JMS75>3.0.CO;2-K

The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl‐p‐aminophenyl derivatives, followed by either matrix‐assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl‐N‐glycosidase F and separated from the proteins by reversed‐phase high‐performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl‐p‐aminophenyl derivative and the samples were further purified by normal‐phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine‐linked complex type composed of mannose‐α1–6‐mannose‐β1–4‐N‐acetylglucosamine‐N‐acetylglucosamine(α1–6‐fucose). This highly degraded structure probably represents the final product of intra‐lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid‐binding protein class to which cerebroside sulfate activator belongs. Copyright © 2000 John Wiley & Sons, Ltd. Abbreviations: CSAct cerebroside sulfate activator protein C8 octyl hydrocarbon chain dHex deoxyhexose ESIMS electrospray ionization mass spectrometry 5‐, 2‐, 1‐ and 0‐CHO 5, 2, 1 and 0‐oligosaccharide containing Fuc fucose GlcNAc N‐acetylglucosamine (Hex)n n hexose units (HexNAc)n n N‐acetylhexose units HPLC high‐performance liquid chromatography HUA human urine CSAct LC/MS liquid chromatography/electrospray ionization mass spectrometry Man mannose MS/MS tandem mass spectrometry NP normal‐phase MALDI matrix‐assisted laser desorption/ionization mass spectrometry P parent ion PNGase peptide N‐glycosidase PKA pig kidney CSAct RP reversed‐phase TFA trifluoroacetic acid TMAPA trimethyl‐p‐aminophenylammonium chloride W/A/F water–acetonitrile–formic acid (50 : 50 : 0.1, v/v).