Deep Proteome Profiling of Circulating Granulocytes Reveals Bactericidal/Permeability-Increasing Protein as a Biomarker for Severe Atherosclerotic Coronary Stenosis

Abstract

Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a two-phase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC-MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC-MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients.

Expanding Proteome Coverage with CHarge Ordered Parallel Ion aNalysis (CHOPIN) Combined with Broad Specificity Proteolysis

“…For instance, ion exchange chromatography (strong cation exchange (SCX) 9−11 and strong anion exchange (SAX) 12,13 ), isoelectric focusing of peptides, 14−16 and high-pH reversed-phase chromatography 17−19 have been used with great success to identify an increasing number of proteins in tissues, 20,21 cells, 22 and other biological samples. 23,24 In addition, complementary digestion using proteases with alternative cleavage specificities can increase protein sequence coverage in deep proteome analyses. 5,25−27 Interestingly, the fragmentation/detection modes also deliver complementary data to increase peptide identification rates.…”

Statistical detection of quantitative protein biomarkers provides insights into signaling networks deregulated in acute myeloid leukemia

“…Alignment-based quantitation of LC-MS/MS experiments can go beyond the rather limited throughput of many proteomic approaches, and due to its additional dimension of separation, as compared with SELDI or MALDI techniques, it is not so prone to the artifacts that have challenged biomarker discovery using these approaches. Therefore, label-free LC-MS/MS is likely to provide a more practical approach to biomarker discovery in leukemia and other complex diseases, in terms of coverage and throughput, toward fulfilling the promises of disease proteomics [21][22][23][24][25][26][27][28][29][30]. For example, global profiling a single sample took 105 min with the Foss et al experimental-computational pipeline [25].…”

“…In particular, LC-MS/MS provides relatively high-quality data with sufficient protein coverage and sensitivity for protein biomarker discovery studies [21][22][23][24][25][26][27][28][29][30]. Toward increased throughput, global protein profiling using label-free LC-MS/MS has received increased interest due to its potential to enable relatively straightforward and comprehensive discovery of quantitative biomarkers in large numbers of clinical samples.…”

Contribution of proteomics to the management of vascular disorders

“…Such techniques include gel based methods like 2-DE, and non-gel based techniques like liquid chromatography tandem mass spectrometry (LC-MS/ MS) and capillary electrophoresis-mass spectrometry (CE-MS). Table 1 identifies different studies in which such techniques were applied in recent years [30,36,44,50,[52][53][54][55][56][57][58][59][60][61].…”