Sensitive and quantitive proteomics and glycoproteomics biological studies




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Proteins and glycoproteins regulate important biological activities, including cell-cell signaling, protein stability, protein localization, and immune response. These biological activities are further associated with human diseases such as inflammatory diseases, rheumatoid arthritis, Alzheimer’s disease, neuronal diseases, and cancer. The new advanced liquid chromatography interfaced to tandem mass spectrometry (LC-MS/MS) methodologies allow proteomics and glycoproteomics analysis that reveals the biological significance of these molecules. On the other hand, to better understand the biological processes, the improvement of analytical methods is needed. In this study, application and development of LC-MS/MS based proteomics and glycoproteomics analysis have been illustrated. Brain injury present a major public health threat affecting nations worldwide. As patients afflicted by brain injury are rising, it is necessary to further investigate the pathophysiological mechanism of this disease. In Chapter 2, the current status and future role of proteomics/glycoproteomics in studies of human neurodegenerative diseases was reviewed. In Chapter 3 and Chapter 4, proteomics/glycoproteomics studies were performed to investigate methamphetamine (METH) induced brain injury and traumatic brain injuries (TBI). The result indicated the expression of 18 proteins (11 in hippocampus and 7 in the olfactory bulb) underwent a significant alteration as a result of exposing rats to METH. The altered proteins belonging to different structural and functional families were involved in processes such as cell death, inflammation, oxidation, and apoptosis. The proteomics/glycoproteomics investigation of two pre-injury antiplatelet therapies: Aspirin (ASA) and Clopidogrel (CLOP), revealed mechanisms associating with unfavorable clinical outcomes of these drugs following TBI. Differential proteins pertaining to each group (22 in TBI, 41 in TBI+ASA, 44 in TBI+CLOP, and 34 in TBI+ASA+CLOP) were identified. Among these proteins, proteins involved in neuroprotective cellular pathways were upregulated in the ASA and CLOP groups when given separately, however, ASA+CLOP administration revealed enrichment in biological pathways relevant to inflammation and pro-injury mechanisms. The glycomics analysis indicated differential upregulation in the sialylated N-glycans, which can be implicated in pathological changes. LC-MS/MS is a high-throughput analytical tools that can be used to identify clinically relevant diagnostic biomarkers. In Chapter 5, sera samples from 205 patients recruited in the U.S. and Egypt were analyzed for biomarker discovery using label-free proteomics analysis by LC-MS/MS. Untargeted proteomic analysis of sera identified candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. The further evaluation using targeted quantitation by multiple reaction monitoring (MRM) revealed the significance of 101 proteins in sera from the same 205 patients. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the U.S. and Egyptian cohorts. Among the 21 candidates, 10 were previously reported as HCC-associated proteins (eight exhibiting consistent trends with our observation), whereas 11 were new candidates discovered by this study. Pathway analysis based on the significant proteins reveals up-regulation of the complement and coagulation cascades pathway and down-regulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. Sample preparation is a key step of proteomics/glycoproteomics analysis since excessive protein debris, slats and detergent will interfere the LC separation and electrospray ionization (ESI). Convenient, efficient and unbiased sample preparation protocols for N-glycan analysis are desired. Recently, one of the commonly used detergents removal strategy -filter aided sample preparations- was successfully introduced to N-glycans sample preparation. In Chapter 6, we evaluated the application of another strategy for detergent removal: using acidic labile detergent in N-glycans sample preparation. Compared with other protocols sodium deoxycholate (SDC) assisted protocol was the most efficient and unbiased. The evaluation of two major SDC removal approaches indicated acidic precipitation was more compatible with N-glycan analysis than ethyl acetate phase transfer. The combination of SDC lysis buffer and beads beating cell disruption demonstrated the use of SDC in sample preparation of N-glycans derived from biological specimens.
The LC separation is extremely important in bottom-up proteomics and glycoproteomics. In Chapter 7, the effect of elution LC gradient on the protein identification was investigated using pooled blood serum. The total number of protein identification number in a certain time frame was shown to be correlated to the water-ACN gradient. To optimize the LC-gradient, several elution gradient patterns were tested. A 2-hour LC gradient with multiple slops was developed to permit the maximal protein identifications. In Chapter 8, we first reported the separation of glycopeptide isomers on porous graphitic carbon (PGC) LC was significantly improved by elevating separation temperature. This finding permitted the isomeric separation of glycopeptides resulting from highly specific enzymatic digestion. After optimizing the experimental conditions such as mobile phase additive and elution gradient, standard glycoproteins bovine ribonuclease B and bovine fetuin was analyzed by PGC-LCMS. Comprehensive structural isomeric separation of glycopeptides were observed and identified by high resolution MS and MS/MS. Both qualitative and quantitative results were consistent with classic proton NMR study. The glycosylation analysis of human alpha-1 acid glycoprotein revealed potential use of PGC-LCMS for complicated glycoprotein analysis in biomarker discovery. High temperature PGC-LCMS was shown as a reliable analytical method providing comprehensive isomeric separation of site-specific glycoprotein analysis. Discovering glycosylation-related markers using existing software is currently not straightforward. Complete characterization of protein glycosylation requires the identification of intact glycopeptides in samples, including identification of the modification site as well as the structure of the attached glycans. In Chapter 9, we presented GlycoSeq, an open-source software tool that implements a heuristic iterated glycan sequencing algorithm coupled with prior knowledge for the automated elucidation of the glycan structure within a glycopeptide from its collision-induced dissociation tandem mass spectrum. GlycoSeq employed rules of glycosidic linkage as defined by synthetic glycan pathways to eliminate improbable glycan structures and build reasonable glycan trees. The software was tested on two sets of tandem mass spectra of N-linked glycopeptides cell lines acquired from breast cancer patients. After employing enzymatic specificity within the N-linked glycan synthetic pathway, the sequencing results of GlycoSeq were highly consistent with the manually curated glycan structures. Hence, GlycoSeq is ready to be used for the characterization of glycan structures in glycopeptides from MS/MS analysis.



Proteomics, Glycoproteomics, Protein Glycosylation, LC-MS/MS