Plasma Fractionation Enriches Post-Myocardial Infarction Samples Prior to Proteomics Analysis

Abstract

Following myocardial infarction (MI), matrix metalloproteinase-9 (MMP-9) levels increase, and MMP-9 deletion improves post-MI remodeling of the left ventricle (LV). We provide here a technical report on plasma-analysis from wild type (WT) and MMP-9 null mice using fractionation and mass-spectrometry-based proteomics. MI was induced by coronary artery ligation in male WT and MMP-9 null mice (4–8 months old; n = 3/genotype). Plasma was collected on days 0 (pre-) and 1 post-MI. Plasma proteins were fractionated and proteins in the lowest (fraction 1) and highest (fraction 12) molecular weight fractions were separated by 1-D SDS-PAGE, digested in-gel with trypsin and analyzed by HPLC-ESI-MS/MS on an Orbitrap Velos. We tried five different fractionation protocols, before reaching an optimized protocol that allowed us to identify over 100 proteins. Serum amyloid A substantially increased post-MI in both genotypes, while alpha-2 macroglobulin increased only in the null samples. In fraction 12, extracellular matrix proteins were observed only post-MI. Interestingly, fibronectin-1, a substrate of MMP-9, was identified at both day 0 and day 1 post-MI in the MMP-9 null mice but was only identified post-MI in the WT mice. In conclusion, plasma fractionation offers an improved depletion-free method to evaluate plasma changes following MI.

The circular relationship between matrix metalloproteinase‐9 and inflammation following myocardial infarction

“…). MMP‐9 levels increase early post‐MI and remain elevated during the first week, mirroring leukocyte infiltration and demonstrating that leukocytes are the primary post‐MI MMP‐9 source .…”

Proteomic analysis of the cardiac extracellular matrix: clinical research applications

“…Similar to tissue proteomics, plasma proteomics has long been burdened by technical issues. The 10 most abundant proteins account for 90% of total protein concentration, making it difficult to identify novel proteins of interest [41–43]. One abundant protein, serum albumin, often has to be removed prior to proteomics analysis [43].…”

“…The 10 most abundant proteins account for 90% of total protein concentration, making it difficult to identify novel proteins of interest [41–43]. One abundant protein, serum albumin, often has to be removed prior to proteomics analysis [43]. There are several commercially available albumin removal kits; most of which are based in immunoaffinity columns.…”

“…Our group has utilized a gel-free plasma fractionation technique using the GelFree 8100 Fractionation System by Protein Discovery, Inc. prior to proteomics analysis. Using this technique, we were able to reduce the presence of highly abundant proteins and enrich samples for the lower abundant ECM proteins [43]. One issue with albumin depletion is this may also result in removal of low abundant cytokines, lipoproteins, and ECM proteins of interest, especially if these proteins are bound to albumin [16].…”

Citrate Synthase Is a NovelIn VivoMatrix Metalloproteinase-9 Substrate That Regulates Mitochondrial Function in the Postmyocardial Infarction Left Ventricle

“…The top 1 cm of the gel, which contained the proteins, was excised and the proteins digested in situ with trypsin. The digests were analyzed by capillary HPLC-ESI-MS/MS on a Thermo Fisher LTQ Orbitrap Velos mass spectrometer, using a top-6 protocol as previously described (14). Mascot (version 2.3.02; Matrix Science) was used to search the MS results against a combination of the mouse subset of the NCBInr database (Mus.…”