Design And Fabrication Of Protein Sample Pretreatment Platform Based On Microfluidic Chip And Imprinted Monolith

Proteomics is the study of protein expression levels,post-translational modifications,and protein-protein interactions on large-scale level,and perform a comprehensive profiling of disease occurrence and life processes at the protein level.It can provide an important technical tool for early diagnosis of disease,drug target discovery,efficacy judgment and prognosis.The pretreatment of protein sample is a necessary process of "bottom-up" proteomics research,which includes operations such as digestion and enrichment.Ubiquitination is one of the most inducible and regulatory post-translational modifications,and it is difficult to detect because of its low abundance.The imprinted monolith has the dual advantages of predetermined recognition of template molecules and high throughput,and can be applied to the online enrichment and separation of target peptides or proteins in complex matrices.Microfluidic chip technology realizes multi-step operations such as reaction and separation of trace samples on the chip through the design of chip pipeline network and microfluidic control.The flexible design and portable integrated functions are expected to provide a rapid reaction and integrated processing method for protein samples.In this paper,a microfluidic chip was designed,on which a trypsin-immobilized enzyme reactor(IMER)was prepared on the surface of poly(trimethylolpropane trimethacrylate)(TRIM)monolith through the "thiol-ene" click chemistry method,and the enzymolysis performance of IMER on the substrate N-α-benzoyl-L-arginine ethyl ester was investigated in detail.Then the online denaturation and digestion performance of the microfluidic chip on bovine serum albumin was investigated.After optimizing the operating conditions,the denaturation and digestion of BSA can be completed within 17 minutes.Chip denaturation-chip IMER,chip denaturation-online IMER and solution denaturation-offline IMER have the same processing capacity for mouse liver samples,obtaining similar protein identification capacity(798,826 vs.843)and better peptide identification capacity(3923,4593 vs.3916).The ubiquitin-modified peptide imprinted monolith was prepared by fragment imprinting method.In the experiment,the specific amino acid sequence "lysineglycine-glycine(KGG)" at the junction of ubiquitin and the modification site of the substrate protein was used as the template to prepared imprinted monolith.The influence of preparation parameters on the adsorption recovery and imprinting effect of the imprinted monolith was investigated,and the maximum imprinting factor of 2.83 was obtained.After optimizing the extraction conditions,the imprinted monolith could specifically enrich and separate KGG from the spiked sample,and the average recovery of 90.8%(RSD = 3.5%)was obtained with 20 consecutive solid phase extractions.The ubiquitin-modified protein enrichment column was prepared by the epitope imprinting monolith technology.In the experiment,the carboxy-terminal nonapeptide of ubiquitin was used as the epitope template to synthesize the surface grafted imprinted monolith by the strategy of the liquid crystal based low crosslinking and chiral molecule doping.The influence of each preparation parameter was investigated in detail.The maximum imprinting factor of 8.1 was obtained at 15% crosslinking degree.The extraction performance of the imprinted monolith on standard sample,spiked sample,and ubiquitin was investigated.Finally,the imprinted monolith was used for the enrichment and separation of proteins in MCF-7 cells,and 4,2 and 7 ubiquitinmodified proteins were identified from the loading effluent,rinsing and eluate,respectively.Compared with 2 ubiquitin-modified proteins through the pentapeptide imprinted monolith adsorbed,the epitope surface grafted nonapeptide imprinted monolith had better protein adsorption capacity.It was proposed for the first time that protein samples were pretreated by protein fractionation/denaturation/digestion/modified peptide enrichment.In the experiment,the polyphenyleneoxyacetate(AP)monolith was selected as the protein prefractionation column.The results showed that the AP monolith could remove 94.6% of MCF-7 cell protein(1.5 mg/m L,30 μL),and 321 proteins and 652 peptides can be newly identified in the eluate.The AP monolith,IMER and KGG imprinted monolith were integrated online through the microfluidic chip.The resulting sample integrated pretreatment method could achieve protein fractionation,denaturation,digestion and peptide enrichment within 9.6 h,and identify 2004 proteins and 8797 peptides from MCF-7 cells,which had 2.8 times and 3.0 times higher identification capacity than untreated samples.For ubiquitination proteomics identification,the number of ubiquitin-modified proteins,peptides and sites identified by this integrated method were 2.0 times higher than that of untreated samples.In summary,this paper designed and fabricated a new protein sample pretreatment method based on microfluidic chip and imprinted monolith,which enhanced the number of protein identification in proteomics research.The chip technology of protein fractionation/denaturation/digestion/peptide enrichment was expected to provide a new sample pretreatment strategy for high-throughput post-translational modification proteomics analysis.