on 05-01-201210:42 AM - edited on 11-09-202101:42 AM by usermigration2
Parker BL, Palmisano G, Edwards AV, White MY, Engholm-Keller K, Lee A, Scott NE, Kolarich D, Hambly BD, Packer NH, Larsen MR, Cordwell SJ. Mol Cell Proteomics. 2011 Aug;10(8):M110.006833. Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia / reperfusion (I/R) injury, is poorly understood. Observation of glycopeptides by mass spectrometry (MS) is challenging due to the presence of abundant, non-glycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide and hydrophilic interaction liquid chromatography (HILIC) with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analysed by liquid chromatography (LC) - MS/MS. This allowed the identification of 1556 non-redundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive 'glycosylations' were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via iTRAQ and validation with dimethyl labeling to analyse changes in glycoproteins from tissue following prolonged I/R (40 mins I / 20 mins R) indicative of myocardial infarction. The iTRAQ approach revealed 80/437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated SPARC and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.