A New Method For High Sensitivity Analysis Of Phosphorylation And Glycation Protein Based On Biomass Mass Spectrometry

Post-translational modifications (PTMs) are the chemical modification of a protein after its translation, and have greatly increased the complexity and the functional diversity of the proteome. Due to the diversity of PTMs, the number of proteome is much greater than the number of the genome. PTMs regulate the activity state, conformation, localization, function of protein and protein-protein interactions. Besides, PTMs play a key role in almost all cellular processes and are closely related with the pathogenesis of many diseases. Consequently, large-scale identification and characterization of PTMs as well as their changes are critical important for the study of cell biology and disease treatment. PTMs specific proteome is an important aspect of the proteomics research. These PTMs include phosphorylation, glycosylation, ubiquitination, methylation, acetylation and so on. Among which, phosphorylation and glycosylation are the most widely studied PTMs.However, the inherent low abundance of phosphopeptides/glycopeptides and the ion suppression effect caused by the co-existence of non-phosphopeptides and non-glycopeptides make the characterization of phosphoproteins/glycoproteins challenging. Technically, the main challenges in studying these post-translationally modified proteins are the development of specific detection and enrichment/purification methods. And the effective protein glycosylation profiling, including the identification of glycoproteins and their glycosylation site as well as the characterization of glycans attached to the glycoproteins. The glycan synthesis is non-template mediated, the structure of glycans is very complicated. Meanwhile, their ionization efficiency and abundance are low. Therefore, the effective release of glycans from glycoproteins, the derivatization as well as the enrichment/purification of the glycans is very important for the MS-based glycan analysis.The research interest of this work focused on the post-translational modification specific proteomics. And the main contribution of this dissertation is to develop a series of novel methods to resolve existing difficulties in specific enrichment and sensitively detection of the phosphoproteins, glycoproteins and glycans. Novel functionalized nanomaterials were developed and effectively applied in phosphopepdies/glycopeptides enrichment, and the feasibility of these methods was validated with the application in the real biological samples. The combination of the derivatization and enrichment method, and the glycan release with Pronase E treatment as well as the corresponding purification method were developled for the glycan analysis. The main content of this dissertation is summarized as follows:In chapter 1, we summaried the development of current proteomics and PTMs specific proteomics as well as their analysis strategies. As phosphorylation and glycosylation are both the critical important PTMs and the challenge for large scale phosphoproteome and glycoproteome research is the development of the effective enrichment strategies. The different techniques and methods for the separation and enrichment, and the applications of functionalized materials for enrichment were summaried in detail. Meanwhile, for the glycan analysis strategy, we outlined the glycan release methods, and the separation/purification methods, then summarized the main chemical derivatization techniques for detection sensitivity improvement. Finally, on this basis, the purpose and meaning of this dissertation were put forward.In chapter 2, mesoporous γ-Fe2O3 material has been designed and synthesized for the phophopeptides enrichment. Designing with two-in-one strategy, the mesoporous γ-Fe2O3 nanocrystal clusters (m-γ-Fe2O3) have been successfully prepared for integrating the functions of effective enrichment and quick separation of phosphopeptides into a single architecture. The obtained m-γ-Fe2O3 have spherical morphology with uniform particle size of about 200 nm and mesoporous structure with the pore diameter of about 9.7 nm, the surface area is as large as 117.8 m2/g. The m-γ-Fe2O3 possessed very high magnetic responsiveness (Ms=78.8 emu/g, magnetic separation time from solution is less than 5s) and were used for the selective enrichment of phosphopeptides. The experiment results demonstrated that the m-γ-Fe2O3 possessed high selectivity for phosphopeptides at a low molar ratio of phosphopeptides/non-phosphopeptides (1:100), high sensitivity (the detection limit was at the fmol level), high enrichment recovery (as high as 89.4%). Moreover, this material is also applied for enrichment of phosphopeptides from the real sample (drinking milk), showing great potential in the practical application.In chapter 3, the silver nanoparticles (Ag-NPs) encapsulated magnetic composite material MCNC@PMAA@Ag-NPs has been designed and synthesized for the glycopeptides enrichment. The composite microspheres were deliberately designed to be constructed with a high-magnetic-response magnetic colloid nanocrystal cluster (MCNC) core, a poly(methacrylic acid) (PMAA) interim layer and a Ag-NPs functional shell with high coverage. Taking advantage of the multiple interactions between glycopeptides and Ag-NPs and the high magnetic susceptibility of the magnetite core, the MCNC@PMAA@Ag-NPs microspheres possess remarkable selectivity for glycopeptides even at a low molar ratio of glycopeptides/non-glycopeptides (1:100) with a rapid enrichment speed (only need 1 min) and simple operation procedure using magnetic separation. Appling this approach,127 unique glycopeptides mapped to 51 different glycoproteins were identified from only 1μL rat serum samples. These results clearly demonstrate that the MCNC@PMAA@Ag-NPs have great potential for purifying and identifying the low-abundant glycopeptides in complex biological samples.In chapter 4, a new method for the selective and sensitive mass spectrometric analysis of glycans was developed by the combination of fluorinated carbon tags derivatization and fluorinated solid-phase extraction (FSPE). The glycans were derivatized using hydrophobic fluorinated carbon tags for the enhancement of glycans ionization, and then the fluorous tagged glycans were enriched via FSPE based on fluorine-fluorine interaction affinity. When using maltoheptaose as a model glycan, the signal intensity of the derivatized glycans is about 30 to 40 fold increased in MALDI-TOF-MS analysis. By virtue of FSPE enrichment, fluorous-tagged glycans could be well enriched and detected from low abundant fluorous-tagged glycans solutions(as low as 0.1 pmol/μL), and from the high contaminated solutions (6.2 M NaCl,2.6 M. NH4HCO3 or 2 M urea). Besides, FSPE could be used for the selective enrichment of fluorous-tagged glycans even with the existence of large amounts of proteins and obvious signal enhancement is achieved compared with the commonly used graphitized carbon-based enrichment method. With the introduction of the fluorous derivatization reagents, the combination of ionization enhancement and glycan purification is realized. Then this strategy was used for the analysis of glycans released from the glycoproteins. In contrast to the traditional prior glycan separation, glycans released from the glycoproteins were derivatized directly, then the fluorous tagged glycans could be well enriched from the contaminated solutions via FSPE and further detected by MS.In chapter 5, we introduce the Pronase E digestion method for the release of glycans from glycoproteins and the corresponding purification method. Pronase E, a non-sepecific enzyme, hydrolyzes all peptide bonds, but its cleavage efficiency is hindered by the proximity of glycan. Therefore, Pronase E treatment released glycans may contain one to several amino acids (small glycopeptides). For the purification of the Pronase E released glycopeptides, our results demonstrated porous graphite carbon (PGC) solid phase extraction was not effective enough. And the purication of the glycopeptides from Pronase E treated mixture could be achieved by the combination of Sep-Pak C18 SPE and PGC SPE. Under the optimized digestion conditions that the ratio of Pronase E to glycoprotein was 4:1, the digestion time was 48h, or the ratio of Pronase E to glycoprotein was 8:1, the digestion time was 24h, and after purification, Asn-linked glycans were obtained. Pronase E digestion provides a new way for the release of glycans from glycoproteins. The released glycans can be used for structure analysis and the residue amino acid can be applied for the glycan derivatization. Further qualitative and quantitative research strategy for glycan analysis can be developed based on this method.