Sensitive analysis of proteins and glycoproteins derived from biological samples: Quantitative proteomics and glycoproteomics by LC-MS/MS



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Proteins are known to play important roles in biological activities. Many of the biological functions of proteins are modulated by protein post-translational modifications (PTMs). Glycosylation is one of the most prevalent protein PTMs, which play many important biological roles such as cell recognition and adhesion, protein stability and localization, and immune response. Moreover, in recent years, the correlation between aberrant glycoproteins and many diseases has been reported. Hence, qualitative and quantitative analysis of proteins and glycans attached is essential to understand biological processes and the development of diseases. Varieties of analytical techniques have been explored to address the analytical needs. Among the different techniques, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is considered as a powerful approach, thus being commonly used. In this dissertation, the development and application of LC-MS-based proteomics and glycoproteomics approaches is illustrated, providing insight into human diseases from the perspective of altered protein expression and glycosylation. Despite the importance of glycoproteins, the identification of the glycosylation site and the elucidation of glycan structures attached to each glycosylation site remain challenging because the analysis of glycopeptides with LC-MS/MS is hindered by microheterogeneity, low ionization efficiency and low abundance of glycopeptides. First, a modified MS-based approach improving the efficiency of glycopeptide identification was illustrated. In this study, in-source fragmentation (ISF) was implemented in LC-MS/MS analysis to facilitate the parallel acquisition of peptide backbone sequence and glycan composition information. The identification of glycosylation sites and peptide backbone sequencing were based on Y1 ions (ion of peptide backbone with an N-acetylglucosamine attached) generated by ISF and the further fragmentation of Y1 ions in the subsequent CID MS/MS, while the glycan composition was interpreted through normal CID MS/MS. Besides the optimization of MS approach for characterization, efforts were also devoted to the improvement of quantitative strategies. When electrospray ionization (ESI) is used, the nonlinearity of signal response is a concern. To achieve accurate and concurrent quantitation of glycans/peptides/glycopeptides, we developed a metabolic labeling strategy to incorporate heavy isotopes into glycans and proteins of cell cultures with high efficiency and reproducibility. The application of labeling strategies makes it possible to run multiple samples in a single run. In this way, the deviations resulted from sample preparation and instruments are eliminated. The LC-MS-based proteomics and glycoproteomics was applied to characterize the site-specific glycan patterns of glycoproteins and to comprehensively profile the proteome concerning diseases status. The characterization and quantitation of the mAb reference material was conducted using a typical LC-MS-based glycoproteomics approach, providing a reference dataset for the assessment of newly developed analytical methods. The analysis of mAbs and other biologics are of great interests because protein attributes significantly affect the efficacy and safety of protein drugs. LC-MS/MS analysis was also performed to investigate the role of host cells and amino acid sequences in defining the glycosylation patterns of gp120 glycoproteins, which would benefit the development of glycan targeting neutralizing antibodies. LC-MS/MS is a sensitive and high-throughput analytical approach that can be used to investigate protein alteration in diseases and identify biomarkers for clinical diagnosis and prognosis. We evaluated the protein of exosomes derived from 5 rhabdomyosarcoma (RMS) cell lines. It was revealed that 36 proteins were specific to RMS-exosomes when compared to datasets for other cancer cells, which could be further evaluated as possible novel biomarkers for this tumor. In the study for the proteome of human carotid atherosclerotic plaques in early and advanced lesions, our study identified candidate regulators that interact with and are involved in the expression of proteins altered between early and advanced lesions.



Proteins, Glycoproteins, LC-MS/MS, Proteomics, Glycoproteomics