A Novel Multiple-channel Apparatus For Packing Capillary Chromatographic Columns And Its Preliminary Application

Capillary liquid chromatography-mass spectrometry(LC-MS) has become the most dominating tool for the qualitative and quantitative analysis of peptides, proteins and proteomes of extremely complicated biological samples. The improvement of separation ability by LC and analysis by advanced MS with high resolution are the key factors to the accuracy of protein analysis. It was almost impossible using traditional apparatus to pack capillary columns longer than 50 cm, or columns packed with ODS packing materials which are smaller than 2μm. Furthermore, the performance of 15 cm columns with 3-5μm ODS packing materials packed at different time with traditional apparatus are different, moreover, traditional column packing process is time-consuming as slurry is pumped into columns with N2, which can provide 25 MPa pressure at most. Protein glycosylation is a quite common and important post-translational modification, which plays an indispensable role in protein sorting, trafficking, and recognition of molecular. Frequently, cell surface and body fluids were detected with aberrant glycosylation structure or abnormalities of glycosylation extent in disease and cancer patients. Thus glycotroteins are often referred to as an indicator of pathologic states and biomarkers and therapeutic targets. To discover more potential diagnostic markers as well as therapeutic targets, qualitative and quantitative analysis of glycoproteins are of great importance. However, the low abundance of glycotroteins made the analysis of secreted glycoprotein difficult, glycoproteins should be enriched before direct analysis. In addition, there are various glycans and many glycosites on different glycoproteins, which made the identification of glycan structures as well as glycosites more challenging.This thesis is consists of four chapters. In the first chapter, commonly used methods of protein analysis and methods of capillary chromatographic column packing were introduced. Also, glycoprotein especially N-glycoprotein enrichment and quantification methods were summarized.In the second chapter, a novel multiple channel apparatus for packing capillary chromatographic columns was introduced. It was designed and manufactured for packing six capillary chromatographic columns with close column efficiency across packed columns in this thesis. Briefly, the apparatus consists of a magnetic stirrer, a liquid chromatographic pump and a multiple-channel can. The reagents used for preparing ODS slurry and stirring condition of the magnetic stirrer were optimized in the study. Two batches of capillary chromatographic columns were packed under the optimum condition, and these packed capillary chromatographic columns were evaluated in the terms of peak capacity, sequence coverage, retention time of three peptide ions and column pressure using the tryptic digest of a bovine serum albumin(BSA) and detected by LC-MS in electrospray ionization(ESI) mode. The experimental results showed that six columns packed at the same time had close column efficiency, however, the column efficiency of twelve capillary chromatographic columns packed at two times was significantly different. In addition, there was no significant column difference when packing one or six capillary chromatographic columns at the same time. The multiple-channel apparatus designed is simple, time-saving, and can be applied to pack capillary chromatographic columns with similar column efficiencies, thus it has evident advantage over traditional onechannel apparatus.To overcome the shortcomings of recent analysis methods, in the third chapter, we introduced a novel method for enrichment and quantifying N-glycoprotein simultaneously. N-glycoprotein are proteins linked with carbohydrates at the site of N-X-S/T sequence(X denote any amino acid residue except proline). We combine hydrazine chemistry with magnetic particles to concentrate N-glycoproteins. To prepare the magnetic nanomaterials for enrichment of N-glycoproteins, first, we synthesized Fe3O4, then decorated it with SiO2 and NH2 groups, respectively, after which we coated NH2 group with PO3 group using POCl3, the magnetic nanomaterials coated with PO3 group could be bind with metal elements such as Tb3+ and Tm3+, finally, we removed the extra metal elements with water wash. DOTA-NHS was used in our research to react with NH2NH2 to get the chemical reagent that combined DOTA with hydrazine, which can be conjugated the DOTA structure to the magnetic nanomaterials coated with metal elements we synthesized previously. N-glycoproteins were left with a N-acetylglucosamine on Asn after being treated by endo H, the hydrazine structure on the DOTA-NHNH2 structure was used to bind aldehydes which were converted from diol groups of carbohydrates on glycopeptides. When glycopeptides were immobilized on the magnetic particles, nonglycopeptides were washed away by applying magnetic field to the nanomaterials. Thus N-glycopeptides were enriched and also been labeled by metal element, different samples can be relatively quantified by using different metal elements after MS analysis. Experimental results shows that magnetic nanomaterials coated with metal elements and DOTA-NHNH2 were successfully prepared. In addition, the dimethyl modification of all the amino groups on the peptides can avoid interference with quantitative accuracy, and was of high efficiency. These results can all contribute to enrichment and quantification of N-glycoproteins.In the fourth chapter, the results and meaning of the work done in this thesis was summarized.