Cardiac extracellular proteome profiling and membrane topology analysis using glycoproteomics

Abstract

Purpose Extracellular proteins are easily accessible, which presents a sub-proteome of molecular targets that have high diagnostic and therapeutic potential. Efforts have been made to catalogue the cardiac extracellular matridome and analyze the topology of identified proteins for the design of therapeutic targets. Although many bioinformatics tools have been developed to predict protein topology, topology has been experimentally validated for only a very small portion of membrane proteins. The aim of this study was to use a glycoproteomics and mass spectrometry approach to identify glycoproteins in the extracellular matridome of the infarcted LV and provide experimental evidence for topological determination. Experimental design Glycoproteomics analysis was performed on eight biological replicates of day 7 post-MI samples from wild type mice using solid-phase extraction of glycopeptides, followed by mass spectrometric identification of N-linked glycosylation sites for topology assessment. Results We identified hundreds of glycoproteins and the identified N-glycosylation sites provide novel information on the correct topology for membrane proteins present in the infarct setting. Conclusions and clinical relevance Our data provides the foundation for future studies of the LV infarct extracellular matridome, which may facilitate the discovery of drug targets and biomarkers.

Plasma Glycoproteomics Reveals Sepsis Outcomes Linked to Distinct Proteins in Common Pathways*

“…Compared with whole proteome analysis, glycoproteomics greatly enriches for extracellular proteins (11). The enrichment of extracellular N -linked glycopeptides allows for identification of circulating factors associated with the sepsis response.…”

“…Extracellular proteins, including cell membrane proteins and secreted proteins, are accessible drug targets that hold high potential for diagnosis and therapeutics. Extracellular proteins are more often modified with carbohydrates than intracellular proteins, allowing N-linked glycoproteomic analysis to greatly enrich for the extracellular proteome (10, 11). Because plasma is composed of extracellular proteins, a glycoproteomics approach also lends itself to plasma analysis.…”

LXR/RXR signaling and neutrophil phenotype following myocardial infarction classify sex differences in remodeling

“…Incident heart failure events were formally adjudicated by Jackson Heart Study from January 1, 2005 to December 31, 2012. Participants who did not have an MI before visit 2, had heart failure at or before visit 2, were diagnosed with MI after 2008, or did not have plasma collected at visit 2 were excluded, leaving a final sample size of 60 participants, which were analyzed using glycoproteomics as described previously [15, 19, 76, 77]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [80] partner repository with the dataset identifier PXD009870.…”

Matrix metalloproteinases as input and output signals for post-myocardial infarction remodeling

“…Using a solid phase extraction of glycopeptides (SPEG) with hydrazide chemistry protocol can be performed to identify glycopeptides in WT and MMP null infarct homogenates or cell culture conditioned media. [73, 74] Since most receptors are glycoproteins, this approach eliminates the potential issue of high levels of albumin in the conditioned media, since albumin is not a glycoprotein and will not be isolated or labeled by this approach. This approach is also useful for ECM-focused analysis (matridomics).…”