A New Functional Polymer-patterned Silicon Substrate For On-Plate Selective Enrichment And Purification Of Peptides

Protein phosphorylation/glycosylation,one of the most important and ubiquitous post-translational modification(PTM)proteins,not only creates a significant diversity in proteins,but also has a vital role in regulating many complex biological processes.The identification of phosphorylation/glycosylation sites and quantification of their dynamic changes greatly enhance the understanding of disease status and play an important role in developing new pharmaceutical targets in several diseases including cancer.The matrix assisted laser desorption/ionization time-offlight mass spectrometry(MALDI TOF-TOF MS)technique has emerged as a powerful tool for characterization of phosphorylated/glycosylated proteins and phosphorylation/glycosylation site mapping.However,MS signals of low-abundance phosphorylated/glycosylated species are severely suppressed by abundant nonphosphopeptides/nonglycopeptides in complex biological samples.In addition,poor analyte-matrix co-crystallization influenced by a relatively high concentration of salts,interference from matrix-related ions and other reagents coexisting in solution results in a significant decrease of MS sensitivity.To date,various methods have been developed for selective enrichment and purification of phosphopeptidesglycopeptides prior to MALDI MS analysis.Most of the above-mentioned approaches are performed in solution,which needs several procedures including labor-intensive desalting and centrifugations at high speed.These tedious steps might cause sample loss which negatively affects the quantification accuracy and decreases the detection sensitivity and restricts the application of the method for the detection of trace phosphopeptides/glycopeptides.Firstly,we assembled a circular hydrophobic-hydrophilic-Ti4+ immobilized phosphate polymer is patterned on a silicon wafer.Such a wafer is used as a novel sample support to allow fast selective enrichment,wash-free self-desalting and mass spectroscopy(MS)analysis of phosphopeptides,thanks to the high Ti4+ loading,amount,pure phosphate polymer-Ti4+ interface,and strong hydrophobic-hydrophilic attraction pattern.The detection sensitivity was enhanced to 5 finol·μL-1,wkich was 300 folds compared with what was obtained using the common MALDI plate.Remarkable selectivity for phosphopeptides can be achieved at a molar ratio as low as 1:500 of phosphopeptides(casein digest)/nonphosphopeptides(BSA).High-quality mass spectra can be obtained even in the presence of NaCl(1 M),NH4HCO3(100 mM),or urea(1 M).These microspots were also used to selectively capture phosphopeptides from milk and human serum,which further demonstrated that they were capable of identifying low-abundance phosphopeptides from real complex samples.They provide a low detection limit(5 fmol·μL-1),small sample size,and excellent enrichment and desalting efficiency.Secondly,we also assembled a circular hydrophobic-hydrophilic-PPA-EAPBA is patterned on a silicon wafer.Such a wafer is used as a novel sample support to allow fast selective enrichment,wash-free self-desalting and mass spectroscopy(MS)analysis of glycopeptides,The specific recognition of PPA-EAPBA to glycopeptides was based on a typical borate esterification reaction,in which the boronic group reacted with cis-diols of glycan to form a heterocyclic diester in alkaline solution,and the esters dissociated on switching the medium to acidic pH.The detection sensitivity was enhanced to 118.5 fmol · μL-1,which was 200 folds compared with what was obtained using the common MALDI plate.Remarkable selectivity for phosphopeptides can be achieved at a molar ratio as low as 1:10 of glycopeptides(HRP digest)/nonglycopeptides(BSA).High-quality mass spectra can be obtained even in the presence of NaCl(1 M),NH4HCO3(100 mM),or urea(1 M).Samely,remarkable selectivity for phosphopeptides can be achieved when the human immunoglobulin G(Ig G)and serum enzyme solution(100 times dilution)mixed according to 1:1.Such a method significantly simplifies the analytical procedures,reduces possible sample loss,and is relatively low cost.Therefore,this on-plate patterned technique is very promising in the high-throughput phosphoproteomic research,especially for the detection of tiny amounts of samples.