Preparation Of Novel Functionalized Covalent Organic Frameworks And Their Application In Proteome Research

The bottom-up proteomics based on mass spectrometry(MS)analysis of proteolytic peptides is the primary technique for high-throughput characterization of post-translational modifications on proteins.Efficient and complete digestion of protein samples prior to MS analysis is a key prerequisite for successful proteome profiling.Traditionad in-solution or in-gel digestion is typically performed for 12-16 h,which greatly limits the rapid and high-throughput analysis of the proteome.Protein phosphorylation and glycosylation are two of the most common and important proteins post-translational modifications(PTMs).Due to the extremely low abundance,the phosphopeptides/glycopeptides usually suffer strong interference with highly abundant nonphosphopeptides and nonglycopeptides,resulting in low ionization efficiency and MS responses.Selective enrichment of low-abundance proteins/peptides with PTMs using functionalized materials is crucial for comprehensive and in-depth analysis of the PTM proteome.Based on the application of functionalized covalent organic frameworks(COFs),there are several works as follows.Novel immobilized enzyme reactor:The time-consuming and costly traditional digestion techniques could be effectively ameliorated by an immobilized enzyme reactor via anchoring the proteases to solid supports.The covalent organic framework-coated magnetic graphene was prepared by a two-step solvothermal reaction and used as a novel solid carrier for efficient covalent immobilization of trypsin with a high degree of loading(up to 268 ?g mg-1).Profiting from the advantages of magnetic graphene and covalent organic frameworks,the novel trypsin bioreactor(MG@TpPa-1-trypsin)was successfully applied for enzymatic digestion of standard proteins.After digestion for 5 min using MG@TpPa-1-trypsin,the obtained sequence coverage(89%)for BS A was obviously higher than that by traditional in-solution digestion for 12 h(75%),while the storage stability,substrate dynamic concentration range and reusability of immobilized enzyme significantly increased.Complete digestion could be achieved in as short a time as 2 minutes.For the digestion and identification of proteins extracted from Amygdalus Pedunculata,a total of 2833 proteins were identified with only a 20-min digestion,which was slightly higher than those obtained by 12 h of in-solution digestion(2739 proteins).Novel functionalized material for phosphopeptide enrichment:To date,plenty of functionalized materials for phosphopeptide enrichment prior to MS analysis appear.However,the complicated and tedious synthesis methods of most materials still need to be improved.A novel covalent organic framework-based immobilized metal ion affinity chromatography(IMAC)material(TpPa-2-Ti4+)was prepared simply by direct immobilizing Ti(IV)into TpPa-2 COFs without any extra chelating ligands,and a new flower-shaped Ti4+-IMAC with regular micro-nano hierarchical structure has been observed in the SEM and TEM images.The obtained titanium(?)ion-modified covalent organic frameworks demonstrated low limit of detection(4 fmol)and satisfactory selectivity(?-casein:BSA?1:100)for phosphopeptide capturing from ?-casein.Similarly,18 and 17 phosphopeptides could be easily detected from the tryptic digest of a-casein or the digest mixture of a-casein and BSA(1:50),respectively.For actual complex samples,twelve phosphopeptides were isolated and identified frorm the digest of non-fat milk.Within three independent replicates,a total of 7432 phosphopeptides corresponding to 2632 phosphoproteins were identified from the digest of HeLa cells,and the number of identified phosphopeptides in any single run was significantly higher than those obtained by classic DHB/TiO2 method(2286 phosphopeptides).Novel hydrophilic material for selective enrichment of glyeopeptides:The development of-specific enrichment materials and strategies before MS analysis was a prerequisite for glycoproteome research due to the low abundance of glycopeptides as well as the interference and suppression by nonglycopeptides.The novel sea urchin-type covalent organic frameworks(Fe3O4@TpPa-l)with super-paramagnetic properties were first constructed via in situ growth of TpPa-1 COFs on the surface of magnetic nanoparticles with only two-step solvothermal reaction.The novel composites were successfully applied for the hydrophilic enrichment of N-glycopeptides,a total of 37 and 22 N-glycopeptides could be easily detected from IgG and HRP digests,respectively.An ultralow detection limit(28 fmol),satisfactory selectivity(IgG:BSA=1:100)and high recovery(86.3-94.3%)could be achieved by Fe3O4@TpPa-1.Within three independent replicates,228 N-glycopeptides corresponding to 114 N-glycoproteins could be detected from human serum digests,which is better than those obtained by commercial HILIC materials(195 N-glycopeptides corresponding to 105 N-glycoproteins).