NEW SAMPLE PREPARATION METHODS FOR PROTEOMIC

The complexity of proteomes brings great challenge to the in-depth study on proteomics, due to the great number of proteins in a sample, the wide dynamic change in abundance, different physical and chemical properties, changes in spaces and with time, and so forth. Therefore, it our recent work, great effort has been made on the development of sample preparation methods for proteomic study. To achieve the selective enrichment of target biomolecules, such as proteins or post-translated peptides, various novel materials were synthesized. For hosphopeptides, the specific enrichment was achieved by agnetic nanoparticles with adenosine triphosphate (ATP) as the ligand for Ti immobilization. Compared to other IMAC materials, with such a ligand, great selectivity was obtained for hosphopeptides. Furthermore, to capture glycopeptides, novel boronic acid functionalized core-shell nanoparticles, poly(MBAA-co-MAA)@VPBA, were prepared, which showed promising for profiling the glycosylation site occupancy and corresponding glycan heterogeneity. To fasten the analysis throughput of proteome, on-line proteome digestion with immobilized enzymatic reactors (IMERs) is a good solution. Herein, hydrophilic supports, such as monolithic poly (acrylamide-co-methylenebisacrylamide), monolithic poly (N-acryloxysuccinimide-co-poly (ethylene glycol) diacrylate), and Graphene oxide, were applied, to decrease the non-specific adsorption of 420 proteins or peptides on the matrices. Besides, various enzymes, such as trypsin, chemotrypsin, pepsin and PNGase were immobilized, to achieve high throughput proteome digestion. To avoid the sample loss and contamination, and to achieve high throughput sample on-line preparation, various integrated sample pretreatment devices were designed. Through the integration of a HILIC trap column for glycopeptides enrichment, an SCX trap column for buffer exchange, and an IMER for deglycosylation, the on-line profiling of glycosites could be achieved within 1 h. urthermore, through the integration on on-line protein denaturation, reduction, buffer exchange and digestion, native proteins could be changed to peptides within a few minutes. All these results demonstrate that new sample preparation ethods play important roles to achieve high resolution, high sensitivity and high throughput analysis of proteomes.The complexity of proteomes brings great challenge to the in-depth study on proteomics, due to the great number of proteins in a sample, the wide dynamic change in abundance, different physical and chemical properties, changes in spaces and with time, and so forth. Therefore, it our recent work, great effort has been made on the development of sample preparation methods for proteomic study. To achieve the selective enrichment of target biomolecules, such as proteins or post-translated peptides, various novel materials were synthesized. For hosphopeptides, the specific enrichment was achieved by agnetic nanoparticles with adenosine triphosphate (ATP) as the ligand for Ti immobilization. Compared to other IMAC materials, with such a ligand, great selectivity was obtained for hosphopeptides. Furthermore, to capture glycopeptides, novel boronic acid functionalized core-shell nanoparticles, poly(MBAA-co-MAA)@VPBA, were prepared, which showed promising for profiling the glycosylation site occupancy and corresponding glycan heterogeneity. To fasten the analysis throughput of proteome, on-line proteome digestion with immobilized enzymatic reactors (IMERs) is a good solution. Herein, hydrophilic supports, such as monolithic poly (acrylamide-co-methylenebisacrylamide), monolithic poly (N-acryloxysuccinimide-co-poly (ethylene glycol) diacrylate), and Graphene oxide, were applied, to decrease the non-specific adsorption of 420 proteins or peptides on the matrices. Besides, various enzymes, such as trypsin, chemotrypsin, pepsin and PNGase were immobilized, to achieve high throughput proteome digestion. To avoid the sample loss and contamination, and to achieve high throughput sample on-line preparation, various integrated sample pretreatment devices were designed. Through the integration of a HILIC trap column for glycopeptides enrichment, an SCX trap column for buffer exchange, and an IMER for deglycosylation, the on-line profiling of glycosites could be achieved within 1 h. urthermore, through the integration on on-line protein denaturation, reduction, buffer exchange and digestion, native proteins could be changed to peptides within a few minutes. All these results demonstrate that new sample preparation ethods play important roles to achieve high resolution, high sensitivity and high throughput analysis of proteomes