Development Of Methods For Selective Analysis Of Target Biomolecules By Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry?MALDI-MS?has been widely used for proteomics,biomarker screening and microorganism identification because of its characteristics of simple operation,high throughput,sensitivity,intuitive spectrum,and high salt tolerance.However,the current challenge faced by MALDI-MS technique is the effective ionization of target biomolecules that lack basic groups and possess high polarity or poor stability,and thereby simplifying laborious sample pretreatment to achieve high-throughput analysis.For example,the important biomolecules such as carbohydrates and phosphopeptides have lower ionization efficiency and easily occur dissociation in MALDI-MS.Moreover,with the presence of other compounds that possess high ionization efficiency in MALDI-MS,the signals of these biomolecules are always suppressed.Therefore,enrichment and/or derivatization are always required before MALDI-MS analysis.These pre-processing steps are tedious,time-consuming,and easily lead to loss of sample information.For MALDI-MS analysis,the matrix and sample preparation directly affect the results of qualitative and quantitative analysis.Hence,it would be appealing to develop MALDI-MS methods for selective detection of target biomolecules from complex mixture by modification/design of matrix or introducing ionization tags.Herein,aiming to develop simple,fast,and high-throughput MALDI-MS methods for selective detection of target biomolecules,the following research works have been carried out in this paper.I.In the study of protein phosphorylation,the identification of phosphopeptides by MALDI-MS still has limitations because of its lower ionization efficiency caused by the negatively charged phosphate groups and low abundance in peptide mixtures.Herein,an ionic liquid matrix consisting of 1,1,3,3-tetramethylguanidine?TMG?and2,4,6-trihydroxyacetophenone?THAP?was developed for MALDI-MS to enhance ionization efficiency of phosphopeptide.After a series of optimizations,the ionic liquid matrix with a TMG-to-THAP molar ratio of 3:1 showed the best performance for phosphopeptide analysis;this matrix was denoted as G₃THAP.Furthermore,the addition of phosphoric acid?PA?to G₃THAP significantly reduced the background noise.The resultant matrix denoted as G₃THAP/PA decreased the dissociation of the phosphopeptides and showed great reproducibility in the negative ion mode.Also,G₃THAP/PA selectively enhanced the ionization of phosphopeptides in the presence of high concentrations of salts.Finally,the selective analysis of phosphopeptides in proteolytic digests by MALDI-MS was also demonstrated and compared to the results obtained using another reported ionic liquid matrix 3-aminoquinoline/a-cyano-4-hydroxycinnamic acid/ammonium dihydrogen phosphate?3-AQ/CHCA/ADP?and the commonly used solid matrix 2,5-dihydroxybenzoic acid?DHB?/PA.In general,G₃THAP/PA is a cool and high salt-tolerant ionic liquid matrix for the preferential ionization of phosphopeptides by negative ion MALDI-MS,showing great potential in the study of protein phosphorylation.II.Glycosylation is another important post-translational modification of proteins.Characterization of glycans is of great interest to glycobiology research.However,analysis of glycans by MALDI-MS still remains challenging because of their poor ionization efficiency caused by the low proton affinity and hydrophilic nature of glycans.Moreover,the glycan signal is always suppressed by other compounds with high proton affinity,such as peptides and proteins,in complex samples during MALDI-MS analysis.Aiming to enhance the ionization efficiency of glycans and simplify the sample preparation procedure before MALDI-MS analysis,we reported herein a novel reactive matrix,2-phenyl-3-?p-aminophenyl?acrylonitrile?PAPAN?,for sensitive and selective detection of glycans.PAPAN is a derivative of?-cyanocinnamic acid?CCA?,which possesses high ionization efficiency in MALDI-MS.The PAPAN reacts with the terminal aldehyde of glycans and thereby enables the significant enhancement of ionization efficiency of glycans.As a result,using PAPAN as a reactive matrix,the detection sensitivity for glycans was improved 100-fold compared with that using DHB as the matrix.Also,the sample spot prepared by our optimized spotting method showed excellent reproducibility.Meanwhile,the ionization of peptides could be significantly suppressed using PAPAN as the matrix,which allowed the selective detection of N-glycans from a deglycosylated tryptic digest of glycoprotein without any pretreatment.Moreover,the PAPAN matrix also endowed the analysis of glycans with enhanced fragmentation during MS/MS analysis,which could facilitate glycan structure interpretation.Finally,PAPAN was successfully used for the analysis of N-glycome in human serum.Thus,a simple,sensitive,and selective method for the analysis of glycans has been achieved by using a novel reactive matrix,PAPAN.III.Based on the great performance of PAPAN,a rapid and highly reproducible MALDI-MS method for profiling of oligosaccharides in beer has been reported by employing PAPAN.After a series of optimization including sample dilution factor,types of cationization agent and its concentration,the distributions of maltooligosaccharides in different brands of beers were unambiguously identified.The present method enabled the direct MALDI-MS analysis after incubating the beer sample with PAPAN matrix solution for one hour.Since the PAPAN selectively reacts with the reducing end of oligosaccharides,the interfering substances could be effectively eliminated.Thus,this method shows great potential for the analysis of oligosaccharides in other foods.IV.In recent years,enzyme screening based on MALDI-MS has developed rapidly.However,it is still required to develop methods that selectively detect the specific substrates/products from the enzyme-catalyzed reaction system and improve the reliability of the quantification,simultaneously.Herein,a rationally designed pyrene linked peptide probe for quantitative protease activity assay via MALDI-MS has been reported.In this proof-of-concept study,a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe,Py-GGGGRG.The introduction of pyrene greatly increased ionization efficiency of Py-peptides,and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene?PS?coated MALDI plate through hydrophobic and?-?stacking interactions.As a result,trypsin activity could be directly quantified by relative intensity ratio of product and substrate?Ip/Ip+Is?via MALDI-MS without the use of external internal standard.A linear range of 0.1¹0?g/mL and a relatively low detection limit of 29 ng/mL were obtained.This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors.Besides,the proposed strategy was also validated for another protease,chymotrypsin,by using the probe Py-GGGGGGYG.Therefore,owing to simplicity,high-throughput capacity and quantificational accuracy,the proposed method shows great potential for activity assay of various proteases via application of specific peptide sequences.