Relative Quantitation of Glycoisoforms of Intact Apolipoprotein C3 in Human Plasma by Liquid Chromatography鈥揌igh-Resolution Mass Spectrometry

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• 作者：Wenying Jian ; Richard W. Edom ; Dai Wang ; Naidong Weng ; Stanley (Weihua) Zhang
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：March 5, 2013
• 年：2013
• 卷：85
• 期：5
• 页码：2867-2874
• 全文大小：356K
• 年卷期：v.85,no.5(March 5, 2013)
• ISSN：1520-6882

文摘

Glycosylation is one of the most important post-translational modifications to mammalian proteins. Distribution of different glycoisoforms of certain proteins may reflect disease conditions and, therefore, can potentially be utilized as biomarkers. Apolipoprotein C3 (ApoC3) is one of the many plasma glycoproteins extensively studied for association with disease states. ApoC3 exists in three main glycoisoforms, including ApoC3-1 and ApoC3-2, which contain an O-linked carbohydrate moiety consisting of three and four monosaccharide residues, respectively, and ApoC3-0 that lacks the entire glycosylation chain. Changes in the ratio of different glycoisoforms of ApoC3 have been observed in pathological conditions such as kidney disease, liver disease, and diabetes. They may provide important information for diagnosis, prognosis, and evaluation of therapeutic response for metabolic conditions. In this current work, a liquid chromatography(LC)鈥揾igh-resolution (HR) time-of-flight (TOF) mass spectrometry (MS) method was developed for relative quantitation of different glycoisoforms of intact ApoC3 in human plasma. The samples were processed using a solid-phase extraction (SPE) method and then subjected to LC鈥揻ull scan HRMS analysis. Isotope peaks for each targeted glycoisoform at two charge states were extracted using a window of 50 mDa and integrated into a chromatographic peak. The peak area ratios of ApoC3-1/ApoC3-0 and ApoC3-2/ApoC3-0 were calculated and evaluated for assay performance. The results indicated that the ratio can be determined with excellent reproducibility in multiple subjects. It has also been observed that the ratios remained constant in plasma exposed to room temperature, freeze鈥搕haw cycles, and long-term frozen storage. The method was applied in preliminary biomarker research of diabetes by analyzing plasma samples collected from normal, prediabetic, and diabetic subjects. Significant differences were revealed in the ApoC3-1/ApoC3-0 ratio and in the ApoC3-2/ApoC3-0 ratio among the three groups. The workflow of intact protein analysis using full scan HRMS established in this current work can be potentially extended to relative quantitation of other glycosylated proteins. To our best knowledge, this is the first time that a systematic approach of relative quantitation of targeted intact protein glycoisoforms using LC鈥揗S has been established and utilized in biomarker research.