Search

Because blood interacts with almost all tissues of the body, it is likely that changes in the overall health of an organism will be reflected in the quantities of specific serum peptides and proteins, making them biomarkers. Due to the complexity of serum, pre-analytical sample simplification and separation are needed prior to mass spectrometric analysis. Use of a reverse-phase capillary column coupled to a mass spectrometer allows for separation and analysis of serum as part of efforts to discover biomarkers. Even after sample simplification by organic solvent precipitation, data files for a single sample typically exceed one gigabyte, making it difficult to analyze complete serum mass spectrometry profiles with currently available software. However, with adequate safeguards, it appears possible to consider portions of mass spectra to find differences in peak intensities between clinical comparison groups visually. To facilitate this, the elution profile was divided into 2-min intervals in which mass spectrometry data were averaged. This required that molecular species had defined reproducible elution times. Given liquid chromatography coupled to mass spectrometry variation, misalignment of elution times of individual peaks occurred often. Hence, internal time controls were identified within each window and used for elution time normalization. This significantly reduced variability in data. This approach allowed for peak alignment across samples, improving biomarker discovery.

Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect. Little is known about the variability of complex proteomes in response to the environment. Two methods-two-dimensional gel electrophoresis (2DGE) and capillary liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LCMS)-were used to study the hamburger effect in two cross-sections of the soluble fruit fly proteome. 2DGE measured abundant proteins, whereas LCMS measured small proteins and peptides. Proteomic differences between males and females were first evaluated to assess the discriminatory capability of the methods. Likewise, wild-type and white-eyed flies were analyzed as a further demonstration that genetically based proteomic differences could be observed above the background analytical variation. Then dietary interventions were imposed. Ethanol was added to the diet of some populations without significant proteomic effect. However, after a 24-h fast, proteomic differences were found using LCMS but not 2DGE. Even so, only three of approximately 1000 molecular species were altered significantly, suggesting that the influence of even an extreme diet change produced only modest proteomic variability, and that much of the fruit fly proteome remains relatively constant in response to diet. These experiments suggest that proteomics can be a viable approach to biomarker discovery.

To detect diseases early in the general population, new diagnostic approaches are needed that have adequate sensitivity and specificity. Recent studies have used mass spectrometry to identify a serum proteomic pattern for breast and ovarian cancer. Serum contains 60-80 mg/mL protein, but 57-71% of this is serum albumin, and 8-26% are gamma-globulins. These large proteins must be depleted before smaller, less-abundant proteins can be detected using mass spectrometry, but because serum albumin is known to act as a carrier for smaller proteins, removal of these molecules using columns or filtration may result in the loss of molecules of interest. The objective of this study was to develop a reproducible method to deplete serum samples of high-abundance proteins in order to analyze the less-abundant proteins present in serum. We used organic solvents to precipitate the large proteins out of solution. We also predicted that this would cause many smaller proteins to dissociate from their carrier molecules, allowing for detection of a larger number of peptides and small proteins. These treated samples were analyzed using capillary liquid chromatography coupled with electrospray ionization mass spectrometry. Analysis demonstrated reproducible results. Acetonitrile treatment clearly released many carrier-bound molecular species and was superior to ultrafiltration alone for serum proteomic analysis.

Post-translational modifications are used by cells to control the functions of proteins. Phosducin-like protein (PhLP) is a regulator of G-protein signaling that is post-translationally modified via phosphorylation. Phosphorylation of PhLP initiates its degradation by the 26S proteasome in serum-stimulated cells. In this report, we show that PhLP is phosphorylated in serum-stimulated Chinese hamster ovary (CHO) cells. Through the use of tandem mass spectrometry (MS/MS), the specific amino acids phosphorylated can be identified. A PhLP-myc-His construct was purified and phosphorylated by serum-stimulated CHO extract. The resulting protein was digested with trypsin and the peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Automated collison-induced dissociation data acquisition was compared with LC-MS/MS of manually chosen parents. In general, LC-MS/MS is superior for parent ions chosen manually, with the notable exception that automated fragmentation employs dynamic collision energy, which can result in higher quality collison-induced dissociation. Using the LC-MS/MS methods, four phosphorylation sites on PhLP were positively identified.

Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins.