Design And Synthesis Of Functionalized Porous Nanomaterials And Their Applications In The Proteomic Analysis

Nanoporous materials have highlighted both material science and nanotechnology for decades. In view of their structures and adjustable surface chemistry, porous materials exhibit extraordinary properties when compared with traditional nanoparticles in versatile applications. More recently, porous materials with large pore sizes have been found to be extremely attractive for various open-ended bio-applications, such as sensors, drug delivery and nano-reactors for efficient proteolysis. Macroporous materials with pore diameters above 50 nm have a high amount of immobilization capacity and fast mass-transfer properties for large size biomolecules (e.g., proteins) and their reactions, making macroporous materials ideal candidates for the proteomics research. However, so far there has been little report on using macroporous materials as substrates for protein profiling, especially for the challenging researches of post-translational modifications (PTM) of proteins. The plain chemistry of pure silica may limit the interaction between unmodified silica surface and proteins with specific post-translational modifications. Thus, it is crucial to carefully design functional macroporous materials aimed for the desired PTM research.In chapter 1, we review the synthesis and fabrication of nanoporous materials,, and their bio-applications in target research field. More specifically, some recent progresses on bio-mass spectrometry and proteomics are examined. These progresses include the study on protein post translational modification, immobilized protease for fast proteolysis et al. Based on the above discussions, general concepts of applications based on macroporous materials were brought forward for the mass spectrometry based proteome research and bio-applicationsIn chapter 2, we introduce a novel nanospace-confined digestion strategy by directly adding the advanced macroporous materials (-100 nm in diameter) as catalysts to the conventional in-solution reaction system. Without increasing the enzyme or protein concentrations, this novel digestion approach exhibits high proteolysis efficiency and selectivity due to the in situ adsorption of both enzymes and proteins from solution into the macropores of catalysts. As a result, the target substrates and enzymes are selectively highly concentrated and confined in the nanospace to realize a ultra-fast digestion. Compared to the proteins, the pore sizes of macroporous materials are large enough to assure a very efficient mass transfer process, thereby accelerating the whole reaction rate. Further selective extraction and digestions of proteins with different isoelectric points can be achieved by adjusting the surface charge of the catalysts based on the electrostatic interactions matching between the biomolecules and catalysts. The catalyst assisted reaction system has been successfully applied to the analysis of the complex biological samples, where 293 proteins are identified, while only 100 proteins are obtained by the standard overnight in-solution digestion. This catalyzed digestion strategy will lead to promising advances in current proteome research.In chapter 3, a phospho-directed nanoreactor with multiple functions is fabricated. Alumina functionalized macroporous ordered silica foams (Al-MOSF) have been developed with large pore size (-100 nm in diameter), high pore volume (1.6 cm3/g) and a surface area of 186 m2/g rich in coordination unsaturated Al-species, which can be used as a phospho-directed nanoreactors for integrated in situ nanodigestion and in situ phosphoisolation. By directly adding Al-MOSF to the conventional in-solution protein-enzyme digestion system, both enzymes and proteins are quickly enriched in the macropores of the reactor in situ to achieve an ultra-fast proteolysis without increasing the enzyme/protein concentration or using pre-immobilization process, thus the digestion time and the cost can be greatly reduced. Meanwhile, due to the chemo-affinity between alumina and phosphogroups, the phospho-directed nanoreactor can in situ isolate specific products of the enzymatic reaction (phosphopeptides) and release the non-specific peptides to the solution. Our strategy is simple, efficient, and can be utilized not only in the analysis of standard phosphoproteins, but also successfully applied in the detection of phosphoproteins in bio-samples. In chapter 4, we develop an advanced glycol-directed nanoreactor/device to achieve unique functions in the biomolecules reactions. Through surface modification with boronic acid species to the macroporous ordered silica foams (denoted B-MOSF), ultrafast in situ digestion and selective glycopeptides isolation can be simultaneously conducted in 30 min via the well designed nanoreactors with a detection limit of glycopeptides with 2.5 pmol digest consumed comparable to the current results. The B-MOSF nanoreactor had fine fit pore size (-100 nm) and immobilization kinetics (< 1 min to 95% of the saturated capacity) for both glycoprotein and enzyme. Thus, nanodigestions can be operated in the confined but not crowded nanospace with ultra-high enzymatic kinetics. Meanwhile, as the surfaces of the B-MOSF nanoreactors were rich in the binding sites (boronic acid species), glycopeptides generating from the in situ digestion can be selectively captured onto the surface of the B-MOSF while the non-glycopeptides were released to the solutions. Furthermore, we demonstrated a novel, robust nanodevice based on rational experimental design, aiming at enhanced selectivity utilizing other non-specific macroporous nanoreactors, such as MOSF and NH2-MOSF (MOSF modified by amino groups) for purification. Compared to the B-MOSF nanoreactors, MOSF and NH2-MOSF offered considerable affinities to the non-glycol-products, which reduced the disturbance due to complexity of the digested mixtures. As a result, a highly efficient, sensitive and selective glycol-analysis process can be achieved in the advanced nanodevice for large scale glycoproteomics. It's anticipated that the nanoreactor/nanodevice can not only be employed for proteomics, but also general bio-applications where enzymatic reactions are involved.