Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.
In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.
Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona, Spain. Electronic address: estelagimenez@ub.edu.
Estela
E
Giménez
Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona, Spain.
Meritxell
M
Balmaña
Department of Surgery, Dr. Josep Trueta University Hospital, IdlBGi, 17007 Girona, Spain.
Joan
J
Figueras
Digestive Unit, Dr. Josep Trueta University Hospital, 17007 Girona, Spain.
Esther
E
Fort
Gastroesophagic Cancer Research Group, Research Programme in Cancer, Hospital del Mar Medical Research Institute (IMIM), Dr. Aiguader, 88, 08003 Barcelona, Spain.
Carme de
C
Bolós
Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona, Spain.
Victòria
V
Sanz-Nebot
Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona, Spain.
Rosa
R
Peracaula
Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna, Austria.
Andreas
A
Rizzi
D015415:Biomarkers / Q000097:blood; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007553:Isotope Labeling; D009584:Nitrogen / Q000737:chemistry; D009961:Orosomucoid / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000473:pathology; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D015415:Biomarkers; D009961:Orosomucoid; D011134:Polysaccharides; D009584:Nitrogen
Glycan analysis; Human α(1)-acid-glycoprotein; Mass spectrometry; Pancreatic cancer; Pancreatitis; Zwitterionic hydrophilic interaction liquid chromatography