Abstract
Membrane proteins constitute 20-30 % of the human genome and make up 60 % of all drug targets. They play important roles in key cellular functions such as small molecules transport, cell-cell interactions and cell signaling. Membrane proteins and proteomics have been notoriously difficult to combine. Most proteomic methods commonly used for analysis of soluble proteins cannot be used for membrane proteins, mainly due to their amphipathic nature.
In this thesis, a method is described for quantitative proteomic analysis of membrane protein enriched samples. In paper I the method is applied to small cell lung cancer cell lines to elucidate Doxorubicin resistance mechanisms. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide isoelectric focusing separation is demonstrated by its ability to: i) lowering the complexity of the sample by two thirds while keeping a high proteome coverage (96%), ii) providing high separation efficiency and iii) allowing for peptide validation and possibly identifications of post transcriptional modifications. The work from paper I encouraged us to further explore the properties of narrow range peptide IEF as a separation strategy. We and others have shown that using IEF as the first dimension of separation is a highly suitable method when analyzing complex samples. It provides an orthogonal separation strategy to reversed phase chromatography as well as giving the opportunity to reduce false positives and false negatives generated in the database search, using the pI values of the peptides.
In paper II, we wanted to explore the possibilities of using a combination of narrow range peptide IEF and reversed phase chromatography to increase the information content of a proteomic analysis. In the final two papers of this thesis, the method described in paper I is subsequently applied to clinical material.
In paper III, the membrane protein fractions of benign and malignant adrenocortical tumors are compared. The mitochondrial membrane protein GRIM-19, a negative regulator of STAT3, is identified as down-regulated in the malignant tissue. The possible role of GRIM-19 down-regulation in the tumorigenesis is discussed. In paper IV, the membrane-associated protein population of alveolar macrophages isolated from Sarcoidosis patients is compared with healthy controls. Affected pathways are described and discussed.
