Identification of de novo synthesized and relatively older proteins: Accelerated oxidative damage to de novo synthesized apolipoprotein A-1 in type 1 diabetes

Abdul Jaleel, Gregory C. Henderson, Benjamin J. Madden, Katherine A. Klaus, Dawn M. Morse, Srinivas Gopala, K. Sreekumaran Nair

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

OBJECTIVE - The accumulation of old and damaged proteins likely contributes to complications of diabetes, but currently no methodology is available to measure the relative age of a specific protein alongside assessment of posttranslational modifications (PTM). To accomplish our goal of studying the impact of insulin deficiency and hyperglycemia in type 1 diabetes upon accumulation of old damaged isoforms of plasma apolipoprotein A-1 (ApoA-1), we sought to develop a novel methodology, which is reported here and can also be applied to other specific proteins. RESEARCH DESIGN AND METHODS - To label newly synthesized proteins, [ring-13C6]phenylalanine was intravenously infused for 8 h in type 1 diabetic participants (n = 7) during both insulin treatment and 8 h of insulin deprivation and in nondiabetic participants (n = 7). ApoA-1 isoforms were purified by two-dimensional gel electrophoresis (2DGE) and assessment of protein identity, PTM, and [ring- 13C6]phenylalanine isotopic enrichment (IE) was performed by tandem mass spectrometry. RESULTS - Five isoforms of plasma ApoA-1 were identified by 2DGE including ApoA-1 precursor (pro-ApoA-1) that contained the relatively highest IE, whereas the older forms contained higher degrees of damage (carbonylation, deamidation) and far less IE. In type 1 diabetes, the relative ratio of IE of [ring-13C6]phenylalanine in an older isoform versus pro-ApoA-1 was higher during insulin deprivation, indicating that de novo synthesized pro-ApoA-1 more rapidly accumulated damage, converting to mature ApoA-1. CONCLUSIONS - We developed a mass spectrometry-based methodology to identify the relative age of protein isoforms. The results demonstrated accelerated oxidative damage to plasma ApoA-1, thus offering a potential mechanism underlying the impact of poor glycemic control in type 1 diabetic patients that affects a patient's risk for vascular disease.

Original languageEnglish (US)
Pages (from-to)2366-2374
Number of pages9
JournalDiabetes
Volume59
Issue number10
DOIs
StatePublished - Oct 2010

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

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