A proteomics perspective at human circulating cells

Abstract

This thesis describes both the development and the implementation of novel strategies of mass spectrometry based proteomics used to characterize and investigate human primary circulating immune cells. The studies were performed to gain better insights into protein constituents of these cells and to explore the option of using these cells for obtaining clinically relevant biomarkers. One of these projects involved finding potential biomarkers in circulating cells for coronary artery disease. Contrary to most protein based biomarker studies that target easily accessible patient material which are substantially hampered by dynamic range issues in protein abundance, we investigated circulating cells that should in theory hold disease state specific biochemical information with a more favorable dynamic range. This yielded a total of 2440 quantified proteins of which 62 proteins initially were revealed as significantly different. However upon closer inspection unavoidable and variable contamination of plasma, erythrocyte and leukocyte components became apparent, which could be traced back to the individual patient level. We presented that correlating the quantitative patient/control study with reported quantitative proteomes of potential contaminating cells allows for filtering of false positive biomarkers into the verification phase. Also, a novel method for identifying the platelet releasate was introduced based on a reversed releasate proteomics approach to unambiguously and quantitatively determine the platelets released from activated platelets against a resting platelet background. Using this strategy we were able to quantitatively discriminate the released proteins from uncontrolled lysis products. Monitoring the copy numbers of roughly 4500 platelet proteins it became apparent that following a full stimulation only 124 proteins were significantly released. The released proteins span a concentration range of at least 5 orders of magnitude. Secretion of several of these proteins were confirmed by ELISA analysis. The released proteins were highly enriched in exhibiting the known secretion tags, among them some known high abundant factors. Besides the well-known secreted proteins many novel low abundant proteins could be identified. The proteomes of neutrophil, eosinophil and basophil granulocytes are quantitatively characterized and compared. At present no comprehensive proteomes are available for these 3 granulocytes that are to some extent similar but have diverging roles as well. For each of these cell types a comprehensive proteome was identified and quantified, exposing many novel specific proteins for these three granulocyte types. A first step was taken to find the similarities between neutrophils, eosinophils and basophils, as well as to expose the proteins that may be involved in their diverging roles.