Preparation Of Molecularly Imprinted Mesoporous Materials And The Application In Protein Phosphorylation Analysis

Protein phosphorylation is a widespread post-translational modification of proteins that is involved in many important cellular activities,including cell differentiation,proliferation,signal transduction,molecular recognition,and metabolic processes.In biological samples,phosphorylated proteins/peptides are closely related to the occurrence and development of various diseases,and can be used as important biomarkers for clinical detection.Mass spectrometry has become an important technique for identifying protein phosphorylation due to its high sensitivity and accuracy.However,due to its low abundance,weak ionization efficiency,and the interference of non-phosphorylated peptides,protein phosphorylation presents a huge challenge in mass spectrometry.Therefore,pre-enrichment of phosphorylated proteins /peptides before mass spectrometry identification is particularly necessary.At present,many strategies for enriching phosphorylated peptides can be developed,but most of the strategies tend to preferentially detect monophosphorylated peptides,often ignoring valuable key information contained in multi-phosphopeptides.In the phosphorylation process,although tyrosine phosphorylation has the lowest proportion,it plays an important role in cell signal transduction because it is upstream of the entire phosphorylation process,and it also has important regulatory functions for the occurrence and development of cancer.At present,the commonly used phosphorylated peptide enrichment methods cannot distinguish different phosphorylation forms,the enrichment of tyrosine phosphorylation mainly depends on the strategy of antibodies.Therefore,the development of selective enrichment of phosphorylated peptides has important application significance.Molecular imprinting technology is an important biomimetic technology for simulating enzymes and antibodies.Materials prepared by molecular imprinting technology can specifically identify target molecules,but generally have problems such as slow mass transfer,template leakage and poor binding ability.Mesoporous materials have attracted widespread attention of scientists due to their ordered mesopores,high specific surface area,and strong thermal stability.In this paper,molecularly imprinted technology and mesoporous materials are combined to prepare molecularly imprinted mesoporous materials for the selective recognition and enrichment of phosphorylated peptides,including the following three contents:1.First,we prepared a molecularly imprinted mesoporous material(MIM)using a sol-gel method,using phosphate as a template,APTES as a functional monomer,and tetraethyl orthosilicate(TEOS)as a crosslinking agent,and MALDI-TOF MS was successfully used to identify phosphorylated peptides.The prepared molecularly imprinted mesoporous material has a specific cavity,which can combine the phosphate of the phosphorylated peptide with a functional monomer on the cavity through hydrogen bonding,thereby increasing the ability to selectively enrich the phosphorylated peptide.This method has been successfully used for the selective enrichment of phosphorylated peptides in β-casein hydrolysate and a mixture of β-casein hydrolysate and BSA(mass ratio 1:100)in complex biological samples.2.Based on the preparation of molecularly imprinted mesoporous materials,we used fructose-1,6-diphosphate(FDP)as a template to prepare molecularly imprinted mesoporous materials(multi-MIM)for enrichment of multi-phosphopeptides.FDP contains two phosphates,which can generate more phosphate-binding sites during the preparation process to facilitate binding to multiple phosphates of a multi-phosphopeptides.By optimizing the washing conditions,we have applied this material to the selective recognition of multi-phosphopeptides in β-casein trypsin digest and found that it has a good recognition ability for phosphorylated peptides,proveing that this method can be used to enrich phosphorylated peptides.3.Finally,we used epitope imprinting strategies to prepare molecularly imprinted mesoporous materials using UPTES as a functional monomer and phenylphosphonic acid(PPA)as a template.Phenylphosphonic acid is the "epitope" of phosphorylated tyrosine,which achieves effective binding with phosphorylated tyrosine.UPTES contains a urea group,which can combine with phosphate to form a cyclic hydrogen bond,thereby achieving the purpose of enriching phosphorylated tyrosine.A series of characterizations were performed on the prepared materials,and the concentration of functional monomers was optimized.At the same time,its binding ability and its ability to enrich phosphorylated tyrosine were also studied.The results show that this material has a high specific surface area and can specifically enrich phosphorylated tyrosine from the standard peptide mixture pSer:pTyr = 1:1 and the proteolytic solution pTyr:β-casein = 1:100.In summary,this paper focuses on the enrichment of phosphorylated peptides,and combines molecularly imprinted technology with mesoporous materials to prepare molecularly imprinted mesoporous materials for the selective enrichment of phosphorylated peptides,it provides a new method for the selective enrichment and identification of phosphorylated peptides.