ARTICLE pubs.acs.org/jpr
Amine Metabolomics of Hyperglycemic Endothelial Cells using Capillary LC MS with Isobaric Tagging Wei Yuan,†,‡ Junxiang Zhang§,†,‡ Shuwei Li,†,‡ and James L. Edwards†,‡,* † ‡
Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, Maryland 20850, United States Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States ABSTRACT: Intracellular amine metabolite changes were quantified from hyperglycemic human aortic endothelial cells (HAECs) as a model for macrovascular complications of diabetes. Amines were selectively tagged using the N-hydroxysuccinimide ester (NHS) based isobaric tag DiART (Deuterium isobaric Amine Reactive Tag), synthesized in house. DiART labeling improved chromatographic resolution of derivatized amines, resulted in 100fold signal-to-noise enhancement in mass spectrometry (MS) analyses, and allowed multiplex quantification of four samples concurrently through tandem MS fragmentation. Targeted measurement of 31 DiART-tagged amines demonstrated the limits of detection below 10 nM/100 amol and averaged RSDs less than 5%. Examination of endothelial cells exposed to short-term hyperglycemia resulted in significant changes to alanine, proline, glycine, serine, and glutamine compared to osmotic controls. Discovery of proline elevation in hyperglycemic endothelial cells suggests a role of proline in hyperglycemia-mediated oxidative stress. Exposure of endothelial cells to high glucose for 7 days resulted in reduced cell number and significant changes to 21 amines relative to cell number. Prominent amine elevation from long-term hyperglycemia include aminoadipate as a sign of lysine breakdown through oxidative stress; cystathionine, hypotaurine, and proline indicating an antioxidant response; and glutamine/ glutamate as substrate level activators of additional metabolic pathways. This report is the first investigation of amine changes to hyperglycemic endothelial cells and offers new insights into the pathophysiology of diabetic complications. KEYWORDS: metabolomics, isobaric, amine, diabetes, endothelial, mass spectrometry, diabetic complications
’ INTRODUCTION Diabetes is the leading cause of nontraumatic lower limb amputation, blindness, and end-stage renal failure in the USA.1 In addition, diabetes holds a 3-fold increased risk for macrovascular diseases such as cardiovascular disease and stroke.2 These complications of diabetes are largely correlated to endothelial cell damage and inflammation.3 The risk of developing complications has been related to average blood glucose levels as indicated by HbA1C levels. Increased HbA1C values are strong indicators of developing diabetic complications.4 These studies suggest that metabolism is directly linked to hyperglycemia-mediated endothelial dysfunction. Hyperglycemia causes oxidative stress through inefficiencies of the electron transport chain,5 which in turn leads to endothelial cell damage. Given that diabetic complications are directly linked to glucose metabolism, metabolomic investigations would shed light on the pathogenesis of this devastating disease. While imbalanced glucose metabolism is the major source of complications, hyperglycemia is suspected of altering multiple metabolic pathways including amino acid metabolism. Profiles of amino acids in serum have long been known to be markedly changed in diabetes.6 In fact, amino acids from serum and plasma are currently being investigated as biomarkers for predicting diabetes onset and renal complications.7 9 Specific amines have r 2011 American Chemical Society
been implicated in the pathology of hyperglycemic endothelial cells. For instance, arginine levels have been tied to endothelial vasodilation, and administration of arginine to diabetic animals has shown improvement to nitric oxide dysfunction in endothelium.10 Changes to cysteine-glutathione pathways are also found in diabetic endothelial cells.11 These pathways are directly related to antioxidant mechanisms and therefore perturbed by the hyperglycemia-mediated oxidative stress. Despite these changes, many amine related pathways have been left unanalyzed likely due to a previous lack in sensitivity. Recent advances in analytical technologies now render such investigations possible. Traditional methods of amino acid analysis have used amine derivatization and LC with optical detection. In recent years, analytical systems have been developed to increase sensitivity and selectivity. Through isotope coded tagging of amines, metabolites in urine and serum have been analyzed using HSQC NMR.12 This nonseparation based system allowed for rapid detection and excellent linearity of all amino acids. Separations, though more time-consuming, are easily coupled to MS and therefore offer increased sensitivity. Using capillary Received: August 23, 2011 Published: October 03, 2011 5242
dx.doi.org/10.1021/pr200815c | J. Proteome Res. 2011, 10, 5242–5250
Journal of Proteome Research
ARTICLE
Figure 1. Schematic of DiART labeling of amine metabolites in human aortic endothelial cells (HAECs). Overview of measuring amine metabolites in HAECs by DiART labeling. 1. Endothelial cells are cultured under 1 control: 5 mM glucose and 3 experimental conditions. 2. Samples are rapidly quenched and extracted using cold organic solvent. 3. Each sample is individually labeled with a different DiART reagent, which will yield a unique reporter ion upon MS/MS fragmentation. All samples are mixed to equivalence. 4. Combined sample is analyzed by targeted LC MS/MS. 5. Data is analyzed and quantified by signal intensity.
electrophoresis coupled to MS, metabolite detection limits of
