Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC-MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15-20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains.

Institute of Microbiology v v i Czech Academy of Sciences Videnska 1083 Prague 142 20 Czech Republic

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Kornfeld R.; Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 1985, 54, 631–664. PubMed

Roth J.; Taatjes D. J.; Lucocq J. M.; Weinstein J.; Paulson J. C. Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell 1985, 431287–295. PubMed

Lairson L. L.; Henrissat B.; Davies G. J.; Withers S. G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 2008, 77, 521–555. PubMed

West M. B.; Segu Z. M.; Feasley C. L.; Kang P.; Klouckova I.; Li C.; Novotny M. V.; West C. M.; Mechref Y.; Hanigan M. H. Analysis of site-specific glycosylation of renal and hepatic γ-glutamyl transpeptidase from normal human tissue. J. Biol. Chem. 2010, 2853829511–29524. PubMed PMC

Takeda Y.; Shinzaki S.; Okudo K.; Moriwaki K.; Murata K.; Miyoshi E. Fucosylated haptoglobin is a novel type of cancer biomarker linked to the prognosis after an operation in colorectal cancer. Cancer 2012, 118123036–3043. PubMed

Mehta A. S.; Norton P.; Liang H.; Comunale M. A.; Wang M.; Rodemich-Betesh L.; Koszycki A.; Noda K.; Miyoshi E.; Block T. Increased levels of tetra-antennary N-linked glycan but not core fucosylation are associated with hepatocellular carcinoma tissue. Cancer Epidemiol., Biomarkers Prev. 2012, 216925–933. PubMed PMC

Miyoshi E.; Nakano M. Fucosylated haptoglobin is a novel marker for pancreatic cancer: detailed analyses of oligosaccharide structures. Proteomics 2008, 8163257–3262. PubMed

Anderson N.; Pollacchi A.; Hayes P.; Therapondos G.; Newsome P.; Boyter A.; Smith K. A preliminary evaluation of the differences in the glycosylation of alpha-1-acid glycoprotein between individual liver diseases. Biomed. Chromatogr. 2002, 166365–372. PubMed

Fang M.; Dewaele S.; Zhao Y. P.; Starkel P.; Vanhooren V.; Chen Y. M.; Ji X.; Luo M.; Sun B. M.; Horsmans Y.; Dell A.; Haslam S. M.; Grassi P.; Libert C.; Gao C. F.; Chen C. C. Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat. Mol. Cancer 2010, 9, 215. PubMed PMC

Pompach P.; Brnakova Z.; Sanda M.; Wu J.; Edwards N.; Goldman R. Site specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma. Mol. Cell. Proteomics 2013, 1251281–1293. PubMed PMC

Kaji H.; Ocho M.; Togayachi A.; Kuno A.; Sogabe M.; Ohkura T.; Nozaki H.; Angata T.; Chiba Y.; Ozaki H.; Hirabayashi J.; Tanaka Y.; Mizokami M.; Ikehara Y.; Narimatsu H. Glycoproteomic discovery of serological biomarker candidates for HCV/HBV infection-associated liver fibrosis and hepatocellular carcinoma. J. Proteome. Res. 2013, 1262630–2640. PubMed

Sanda M.; Pompach P.; Brnakova Z.; Wu J.; Makambi K.; Goldman R. Quantitative LC-MS-MRM analysis of site-specific glycoforms of haptoglobin in liver disease. Mol. Cell. Proteomics. 2013, 1251294–1305. PubMed PMC

Comunale M. A.; Rodemich-Betesh L.; Hafner J.; Wang M.; Norton P.; Di Bisceglie A. M.; Block T.; Mehta A. Linkage specific fucosylation of alpha-1-antitrypsin in liver cirrhosis and cancer patients: implications for a biomarker of hepatocellular carcinoma. PLoS One 2010, 58e12419. PubMed PMC

Debruyne E. N.; Vanderschaeghe D.; Van V. H.; Vanhecke A.; Callewaert N.; Delanghe J. R. Diagnostic value of the hemopexin N-glycan profile in hepatocellular carcinoma patients. Physiol. Rev. 2010, 565823–831. PubMed

Mori S.; Aoyagi Y.; Yanagi M.; Suzuki Y.; Asakura H. Serum N-acetylglucosaminyltransferase III activities in hepatocellular carcinoma. J. Gastroenterol. Hepatol. 1998, 136610–619. PubMed

Tsutsumi M.; Wang J. S.; Takada A. Microheterogeneity of serum glycoproteins in alcoholics: is desialo-transferrin the marker of chronic alcohol drinking or alcoholic liver injury?. Alcohol.: Clin. Exp. Res. 1994, 182392–397. PubMed

Campion B.; Leger D.; Wieruszeski J. M.; Montreuil J.; Spik G. Presence of fucosylated triantennary, tetraantennary and pentaantennary glycans in transferrin synthesized by the human hepatocarcinoma cell line Hep G2. Eur. J. Biochem. 1989, 1842405–413. PubMed

Comunale M. A.; Wang M.; Hafner J.; Krakover J.; Rodemich L.; Kopenhaver B.; Long R. E.; Junaidi O.; Bisceglie A. M.; Block T. M.; Mehta A. S. Identification and Development of Fucosylated Glycoproteins as Biomarkers of Primary Hepatocellular Carcinoma. J. Proteome Res. 2009, 82595–602. PubMed PMC

Saldova R.; Royle L.; Radcliffe C. M.; Abd Hamid U. M.; Evans R.; Arnold J. N.; Banks R. E.; Hutson R.; Harvey D. J.; Antrobus R.; Petrescu S. M.; Dwek R. A.; Rudd P. M. Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG. Glycobiology 2007, 17121344–1356. PubMed

Bones J.; Byrne J. C.; O’Donoghue N.; McManus C.; Scaife C.; Boissin H.; Nastase A.; Rudd P. M. Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from host defense response mechanisms. J. Proteome. Res. 2011, 1031246–1265. PubMed

Ashline D. J.; Hanneman A. J.; Zhang H.; Reinhold V. N. Structural documentation of glycan epitopes: sequential mass spectrometry and spectral matching. J. Am. Soc. Mass Spectrom. 2014, 253444–453. PubMed PMC

Alley W. R. Jr.; Madera M.; Mechref Y.; Novotny M. V. Chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated N-linked glycans: a potential methodology for cancer-biomarker discovery. Anal. Chem. 2010, 82125095–5106. PubMed PMC

Kang P.; Mechref Y.; Klouckova I.; Novotny M. V. Solid-phase permethylation of glycans for mass spectrometric analysis. Rapid Commun. Mass Spectrom. 2005, 19233421–3428. PubMed PMC

Pompach P.; Chandler K. B.; Lan R.; Edwards N.; Goldman R. Semi-automated identification of N-Glycopeptides by hydrophilic interaction chromatography, nano-reverse-phase LC-MS/MS, and glycan database search. J. Proteome. Res. 2012, 1131728–1740. PubMed PMC

Chandler K. B.; Pompach P.; Goldman R.; Edwards N. Exploring Site-Specific N-Glycosylation Microheterogeneity of Haptoglobin Using Glycopeptide CID Tandem Mass Spectra and Glycan Database Search. J. Proteome. Res. 2013, 1283652–3666. PubMed PMC

Royle L.; Campbell M. P.; Radcliffe C. M.; White D. M.; Harvey D. J.; Abrahams J. L.; Kim Y. G.; Henry G. W.; Shadick N. A.; Weinblatt M. E.; Lee D. M.; Rudd P. M.; Dwek R. A. HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal. Biochem. 2008, 37611–12. PubMed

Koide T.; Odani S. Histidine-rich glycoprotein is evolutionarily related to the cystatin superfamily. Presence of two cystatin domains in the N-terminal region. FEBS Lett. 1987, 216117–21. PubMed

Delanghe J. R.; Langlois M. R. Hemopexin: a review of biological aspects and the role in laboratory medicine. Clin. Chim. Acta 2001, 3121–213–23. PubMed

Wuhrer M.; Koeleman C. A.; Hokke C. H.; Deelder A. M. Mass spectrometry of proton adducts of fucosylated N-glycans: fucose transfer between antennae gives rise to misleading fragments. Rapid Commun. Mass Spectrom. 2006, 20111747–1754. PubMed

Kellermann J.; Lottspeich F.; Henschen A.; Muller-Esterl W. Completion of the primary structure of human high-molecular-mass kininogen. The amino acid sequence of the entire heavy chain and evidence for its evolution by gene triplication. Eur. J. Biochem. 1986, 1542471–478. PubMed

Lottspeich F.; Kellermann J.; Henschen A.; Foertsch B.; Muller-Esterl W. The amino acid sequence of the light chain of human high-molecular-mass kininogen. Eur. J. Biochem. 1985, 1522307–314. PubMed

Dempsey E.; Rudd P. M. Acute phase glycoproteins: bystanders or participants in carcinogenesis?. Ann. N.Y. Acad. Sci. 2012, 1253, 122–132. PubMed

Whaley K.; Ruddy S. Modulation of the alternative complement pathways by beta 1 H globulin. J. Exp. Med. 1976, 14451147–1163. PubMed PMC

Ripoche J.; Day A. J.; Harris T. J.; Sim R. B. The complete amino acid sequence of human complement factor H. Biochem. J. 1988, 2492593–602. PubMed PMC

Aebi M. N-linked protein glycosylation in the ER. Biochim. Biophys. Acta 2013, 1833112430–2437. PubMed

Parodi A. J. Protein glucosylation and its role in protein folding. Annu. Rev. Biochem. 2000, 69, 69–93. PubMed

Thaysen-Andersen M.; Packer N. H. Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching. Glycobiology 2012, 22111440–1452. PubMed

Comparison of Different HILIC Stationary Phases in the Separation of Hemopexin and Immunoglobulin G Glycopeptides and Their Isomers

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring