title: Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms of haptoglobin in liver disease.
abstract: Development of liver disease is associated with the appearance of multiply fucosylated glycoforms of haptoglobin. To analyze the disease-related haptoglobin glycoforms in liver cirrhosis and hepatocellular carcinoma, we have optimized an LC-MS-multiple reaction monitoring (MRM) workflow for glycopeptide quantification. The final quantitative analysis included 24 site-specific glycoforms generated by treatment of a tryptic digest of haptoglobin with α(2-3,6,8)-neuraminidase and β(1-4)-galactosidase. The combination of LC-MS-MRM with exoglycosidase digests allowed resolution of isobaric glycoforms of the haptoglobin-T3 glycopeptide for quantification of the multiply fucosylated Lewis Y-containing glycoforms we have identified in the context of liver disease. Fourteen multiply fucosylated glycoforms of the 20 examined increased significantly in the liver disease group compared with healthy controls with an average 5-fold increase in intensity (p < 0.05). At the same time, two tri-antennary glycoforms without fucoses did not increase in the liver disease group, and two tetra-antennary glycoforms without fucoses showed a marginal increase (at most 40%) in intensity. Our analysis of 30 individual patient samples (10 healthy controls, 10 cirrhosis patients, and 10 hepatocellular carcinoma patients) showed that these glycoforms were substantially increased in a small subgroup of liver disease patients but did not significantly differ between the groups of hepatocellular carcinoma and cirrhosis patients. The tri- and tetra-antennary singly fucosylated glycoforms are associated with a MELD score and low platelet counts (p < 0.05). The exoglycosidase-assisted LC-MS-MRM workflow, optimized for the quantification of fucosylated glycoforms of haptoglobin, can be used for quantification of these glycoforms on other glycopeptides with appropriate analytical behavior.
authors: Department of Oncology, Georgetown University, Washington, DC 20057, USA.
authors: Miloslav
authors: M
authors: Sanda
authors: Petr
authors: P
authors: Pompach
authors: Zuzana
authors: Z
authors: Brnakova
authors: Jing
authors: J
authors: Wu
authors: Kepher
authors: K
authors: Makambi
authors: Radoslav
authors: R
authors: Goldman
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006528:Carcinoma, Hepatocellular / Q000097:blood*; D016022:Case-Control Studies; D056148:Chromatography, Reverse-Phase; D005260:Female; D005643:Fucose / Q000737:chemistry / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006242:Haptoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008103:Liver Cirrhosis / Q000097:blood*; D008113:Liver Neoplasms / Q000097:blood*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D018709:Statistics, Nonparametric; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D006242:Haptoglobins; D005643:Fucose
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