New Technologies And Methodologies Developed For Protein Separation And Enrichment

There are three main academic values of this doctoral thesis. First, we have developed a new method for enriching and desalting low-abundant proteins based on three kinds of newly designed core-shell nanobeads subsequently. Second, we have designed core-shell boronic-acid functionalized nanoparticles for selectively enriching glycopeptides, followed by multistage MS to characterize glycosylation sites and glycan structures simultaneously. Third, we have established a new mass spectrometry based analysis strategy for high-molecular-weight (HMW) proteome research, which contains a new gel enhanced electrophoresis separation and core-shell nanobeads assisted identification.Nowadays, three challenges in proteomic have been faced.First of all is low-abundant protein research. In the clinical proteome research, disease associated proteins, which would be biomarkers, expressed commonly in low abundance. Identification of low-abundant proteins by mass spectrometry (MS) is still a challenge due to the sample loss and contaminants interference with MS during the sample pretreatment. Several types of nanomaterials, which have been developed to enrich and desalt peptides/proteins from mixtures, still have some drawbacks in practical applications.The second challenge is analyzing glycoprotein. Protein glycosylation, as one of the most important posttranslational modifications (PTMs), regulates cellular mechanisms, including cell adhesion, receptor activation, signal transduction, molecular trafficking and clearance, and endocytosis. Many diseases are caused by glycosylation or glycosidase deficiencies, such as autoimmune disorders, infantile-onset symptomatic epilepsy. In fact, the US Food and Drug Administration has agreed that over half of the recent cancer biomarkers are glycoproteins. To identify glycosylation sites, as well as the primary structures of the glycans, mass spectrometric strategies have been developed. However, as glycopeptides are always low in abundance when the glycoprotein is digested into peptides, the MS signals of nonglycosylated peptides always heavily interfere with those of glycopeptides. Moreover, determination of glycan structures is usually difficult because of their vast diversity. Therefore, selective enriching methods followed by multistage MS technique are necessary for glycopeptide analysis. Last but not the least, high-molecular-weight (HMW) proteome research is still a challenge. Proteins with the molecular weights above 100 kDa, which are commonly defined as HMW proteins, are known to be involved in a number of human diseases and some of them have been approved as cancer biomarkers, such as CA125 for monitoring ovarian cancer in serum, HMW CEA and mucin for monitoring bladder cancer in urine. One bottleneck is lack of highly efficient separation of HMW proteins from complex real samples. Low success rate of identification is the following problem for the HMW proteome analysis. The MS signals could be suppressed by the contaminants due to the long polypeptide chains of HMW proteins, which would be easily attacked by inorganic salts, chaotropes and detergents during separation procedure. Besides, a large number of hydrophobic peptides of the HMW proteins make their ionization in matrix-assisted laser desorption/ionization (MALDI)-systems difficult by using the commercial organic matrices, such as a-cyano-4-hydroxycinnamic acid (CHCA). Therefore, extra two steps for HMW protein identification are needed, including desalting and signal enhancement.In this thesis, we introduce our researches in five chapters.In the first chapter, we give a brief introduction on the proceedings and challenges of proteomics and its related technolegies. Furthermore, we review the nanomaterials and their applications in proteomics. All of the background information provides theotical and applicable support on doing researches.In the second and third chapters, we introduce a developed method based on three kinds of core-shell nanobeads, which were designed for enriching and desalting low-abundant proteins with low limits of detection, rapid enrichment, good reproducibility, high recovery, and powerful desalting ability. These core-shell nanobeads were synthesized without using surfactants. For the SnO2@poly (methyl methacrylate) (PMMA) and TiO2@PMMA nanobeads synthesis, SnO2 and TiO2 nanoparticles modified with poly (ethylene glycol) methyl ether (PEGME) were synthesized by solvothermal methods to prevent them from aggregation firstly, hydroxy-exchange reactions then took place on the PEGME-SnO2 and PEGME-TiO2 surfaces, and MMA monomers were simultaneously polymerized in boiling water to form PMMA beads. After the two nanobeads enrichment procedure, the MS signal intensities and the corresponding signal-to-noise (S/N) ratios of the intact horse heart myoglobin (MYO,400 fmol/μL) were increased by one order of magnitude, while those of the digested MYO (1 fmol/μL) were enhanced by three orders of magnitude. For the ZnO@PMMA nanobeads, the difference from the uppers in synthesis was that ZnO-cores had been prepared through polymerization of methyl methacrylate initiated by the inherent free radicals on the ZnO surface. These optimized ZnO@PMMA nanobeads displayed more powerful enriching and desalting abilities:-80% bovine serum albumin digests were enriched by ZnO@PMMA from 100 amol/μL solution within 10-min incubation; high-quality mass spectra were obtained, even with the presence of saturated NaCl (6.2 M), saturated NH4HCO3 (2.6 M), or 1 M urea. This method was successfully applied to human colorectal cancer proteome research, and eight new proteins have been found.In the fourth chapter, we introduce a new mass spectrometry based analysis strategy has been established here for high-molecular-weight (HMW) proteome research. First, a 2-hydroxyethyl agarose/polyacrylamide (HEAG/PAM) electrophoresis gel was designed for the first time to realize an easy-handling separation method with high spatial resolution for HMW proteins, good reproducibility and mass spectrometry-compatible sliver staining. Second, ZnO@PMMA nanobeads were applied here for enriching and desalting the peptides from the HMW proteins. Third, the peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with the presence of the ZnO@PMMA nanobeads, and their MS signals were enhanced markedly. The success rate of identification for HMW proteins was significantly increased due to high enriching efficiency and salt tolerance capability as well as signal enhancing capability of the ZnO@PMMA nanobeads. We believe that this analysis strategy will inspire and accelerate the HMW proteome studies.In the fifth chapter, we introduce core-shell boronic-acid functionalized nanoparticles SnO2@Poly(HEMA-co-St-co-VPBA) designed for selectively enriching glycopeptides, followed by multistage MS analysis. Such 60-nm sized core-shell nanoparticles are prepared bv means of copolymerization between 2-hydroxyethyl methacrylate (HEMA) grafted on SnO2 nanoparticles, styrene and 4-vinylphenylboronic acid (VPBA). All of the synthesis procedures are completed within 3 h. Cyclic boronate esters form between boronic-acid groups on the polymer chains and cis-diol groups on glycopeptides, and thus almost all intact glycopeptides from low-abundant horseradish peroxidase (HRP) and bovine asialofetuin (ASF) are enriched with high selectivity and efficiency. After enrichment, both intact N-and O-glycopeptides are characterized by multistage MS. Furthermore, we successfully apply this method to the human serum sample for characterizing the target glycoproteins haptoglobin and alpha-1-acid-glycoprotein. The present selective enriching method followed by multistage-MS analysis is proved to be a good choice for routine glycopeptide characterization.