Sample preparation and mass spectrometry in proteome studies

Abstract

Proteome studies aim to link gene to function. Instead of screening for function in a specific protein as in classical protein chemistry, proteins are now screened against a specific phenotype, disease or metabolic state. To meet the workload of large-scale protein identification and characterization, preparative and analytical methods with high throughput, readiness for automation, and a broad dynamic range are constantly needed.

In this thesis, a novel microfluidic compact disc (CD) system was evaluated and applied to biological samples. The CD system offers sample cleanup, enrichment and deposition on a matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) target area. Each CD contains 96 microcolumns. Depending on the application, the columns contain different stationary phases. Both a reverse phase chromatography (RPC) stationary phase and an immobilized metal affinity chromatography (IMAC) resin were studied. The two types of resin are used with tryptic peptides, generated by digestion of proteins. In RPC, the peptides are desalted and the MALDI mass spectra are then used for peptide mass fingerprinting to identify the digested protein(s). In IMAC, phosphopeptides are captured on the column.

The RPC CD was first evaluated against in-solution and in-gel digested bovine serum albumin (BSA) to evaluate the reproducibility and sensitivity of the system. BSA was routinely identified at a sensitivity of 200 amol. A crude protein mixture from endothelial cells was separated by 2D gel electrophoresis and protein spots were further in-gel digested with trypsin. The peptide mixtures were either applied to the RPC CD or desalted by ordinary C18 solid phase extraction. Out of 48 spots, 45 were successfully identified with the CD approach while 24 were identified with C18 solid phase extraction.

The RPC CD was also used to screen for tumor markers in colon cancer and prostatic zone-specific proteins. Proteins upregulated in colon tumor tissue were mostly enzymes and chaperones while the structural proteins constituted a major part of the downregulated proteins identified. Tumors in the prostate usually stem from the peripheral and transition zones. Protein expression, as visualized by twodimensional (2D) gel electrophoresis, differed between the central zone and the peripheral zone together with the transition zone. Notable was the upregulation of peroxiredoxin 2 in the central zone.

The IMAC CD was evaluated against defined phosphopeptides in a background of tryptic BSA fragments. The success rate of the phosphopeptide detection was over 90 percent. The IMAC CD system has also been successfully applied to gel-separated phosphoproteins and biological samples. Gel-separated phosphoproteins were detected on the IMAC CD down to a level of I pmol protein applied to SDS PAGE. Applied to non-characterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.

Characterization of post-translationally modified proteins was further studied for a novel aldo-keto reductase (AKR) and myelin basic protein (MBP). The AKR was found to be N-terminally acetylated while a novel phosphorylation site at threonine 94 was located in the MBP.