MSACL 2019 EU Abstract

The Analysis of Alpha-1-Antitrypsin Glycosylation in HCC Patient Serum

Haidi Yin1*, Jianhui Zhu2, David M. Lubman2, and Zhongping Yao1
1. The Hong Kong Polytechnic University, Department of Applied Biology and Chemical Technology, Hong Kong; 2. University of Michigan Medical Center, Department of Surgery, Ann Arbor, MI 48109, USA

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Haidi Yin (Presenter) The Hong Kong Polytechnic University

Presenter Bio: Dr. Yin Haidi

The Hong Kong Polytechnic University
Department of Applied Biology and Chemical Technology

RESEARCH BACKGROUND AND RESEARCH INTEREST
* The qualitative and quantitative analysis of proteins and its post translational modification glycosylation using mass spectrometers as well as other biological/analytical methods, biomarker discovery of various diseases

EDUCATION
* Postdoctoral study, Research field: liver cancer marker screening, proteomics
University of Michigan Medical School, Ann Arbor, USA, 09/2012-08/2015
* Exchange student
Institute of Molecular Phytopathology and Mycotoxin Research,
Goettingen University, Goettingen, Germany, 3/2010-12/2010
* Ph.D of Zoology, Research field: Biochemistry and Molecular Biology
College of Life Sciences, Peking University, Beijing, China, 09/2005-07/2011

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction:
The change in glycosylation of some serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. The traditional immunoassay-based method relies on a specific antibody, the quality of which is not stable from batch to batch, resulting in inaccurate qualification and quantification. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate qualification and quantification of a serum protein alpha-1-antitrypsin (A1AT) in early stage HCC and cirrhosis patient serum.

Methods:
Serum protein A1AT was purified from patient sera with anti-A1AT antibody conjugated agarose beads. Isolated A1AT protein was digested and analyzed by LC-MS/MS. The stepped HCD strategy enables the qualification of glycopeptides of A1AT and the parallel reaction monitoring (PRM) strategy is used to quantify various glycoforms of A1AT glycopeptides in HCC and cirrhosis patient sera. pGlyco2.0 software was used for glycan identification and skyline software was used for glycoform quantification using Y1 ion in MS/MS spectrum.

Results:
In-source dissociation was found to severely affect the identification and quantification of glycopeptides. Therefore, the settings of the mass spectrometer were optimized to minimize the in-source dissociation of glycopeptides, including RF lens, the temperature of ion transfer tube, and capillary voltage.
We identified 11 glycopeptides A1AT with stepped HCD, 7 of which were quantified using PRM among patient samples. We found PRM strategy was able to distinguish several isomers of the glycopeptide. Several isomers showed specific pattern in some HCC patients. We also found that the ratio of different charge states of one glycopeptide of A1AT can significantly discriminate early stage HCC from cirrhosis.

Novel Aspect:
This methodology can be applied to identify and quantify glycosylation of any key glycoprotein in various diseases.