Development Of Matrix-assisted Laser Desorption Ionization Mass Spectrometry Techniques For Rapid And Highly Sensitive Detection Of Biological Samples

Matrix-assisted laser desorption ionization mass spectrometry?MALDI-MS?nowadays has become an essential analytical tool for proteomics.Despite its widespread use,there still have many challenges and shortcomings in the actual sample analysis work,as follows:I.Some disease-associated marker proteins are often low-expressed in vivo,and the use of MS to detect low abundant proteins is always a great challenge.II.MALDI-MS has lower ionization efficiency toward some analytes?e.g.,membrane proteins?.III.High concentrations of salts,chaotropes,or surfactants in biological samples often interfere with MS analysis,resulting in poor spectral reproducibility and detection sensitivity.IV.Some sample pretreatments tend to be cumbersome and costly,and have been limited in high-throughput analysis.V.MALDI-MS has large background interference in the small molecule region,which cannot meet the analytical requirements for the spectral identification and sensitivity in the low m/z region.VI.Analytes are difficult to be quantified in MALDI-MS.Against the abovementioned issues,the corresponding solutions were proposed through literature research.This paper starts from the research and development of MALDI organic small molecule matrix,the surface modification on MALDI target plate and the probe design of protease.1.Herein,aseriesofcarboxyl-esterifiedderivativesof?-cyano-4-hydroxycinnamic acid?CHCA?were synthesized and evaluated as matrices for MALDI-MS analysis of protein.Among them,?E?-propyl?-cyano-4-hydroxyl cinnamylate?CHCA-C3?was found to exhibit excellent assay performance for intact proteins by improving the detection sensitivity 10 folds compared with the traditional matrices[i.e.,super2,5-dihydroxybenzoic acid?superDHB?,sinapic acid?SA?,and CHCA].In addition,CHCA-C3 was shown to have high tolerance to salts,the ion signal of myoglobin was readily detected even in the presence of urea?8 M?,NH₄HCO₃?2 M?,and KH₂PO₄?500 mM?,meanwhile sample washability was robust.These achievements were mainly attributed to improved ablation ability and increased hydrophobicity or affinity of CHCA-C3 to proteins in comparison with hydrophilic matrixes,leading to more efficient ionization of analyte.Furthermore,direct analysis of proteins from crude egg white demonstrated that CHCA-C3 was a highly efficient matrix for the analysis of low abundance proteins in complex biological samples.These outstanding performances indicate the tremendous potential use of CHCA-C3 in protein profiling by MALDI-MS.2.Despite the significance of membrane proteins?MPs?in biological system is indisputable,their specific natures make them notoriously difficult to be analyzed.Particularly,the widely used MALDI-MS prefers analyses of hydrophilic cytosolic proteins and has a limited ionization efficiency towards hydrophobic MPs.Herein,a hydrophobic compound CHCA-C3 was applied as a contaminant tolerant matrix for high sensitivity MALDI-MS analyses of MPs.With CHCA-C3,the detection limits of hydrophobic peptides were 10-to 100-fold better than those using CHCA.Furthermore,high quality of spectra could be achieved in the presence of high concentration of chaotropes,salts and detergents,as well as human urinary and serum environment.Also,CHCA-C3 could generate uniform sample distribution even in the presence of contaminants.This high contaminant-resistance was revealed to be ascribed to the enhanced hydrophobicity of CHCA-C3 with a lower affinity towards hydrophilic contaminants.The application of CHCA-C3 is further demonstrated by the analysis of trypsin/CNBr digests of bacteriorhodopsin containing seven transmembrane domains?TMDs?,which dramatically increased numbers of identified hydrophobic peptides in TMDs and sequence coverage?¹00%?.Besides,a combined method by using CHCA-C3 with fluoride solvent and a patterned paraffin plate was established for analysis of integral MPs.We achieved a low detection limit of 10 fmol for integral bacteriorhodopsin,which could not be detected using traditional matrices such as 3,5-dimethoxy-4-hydroxycinamic acid,2,5-dihydroxyacetophenone even at sample concentration of 10 pmol.3.We report a novel strategy to achieve simultaneous one-step in-situ selfdesalting and enrichment?OISE?of peptides/proteins on a facilely fabricated patterned MALDI steel plate with a circular paraffin-steel-polystyrene structure.The OISE plate could efficiently segregate salts from both analytes and matrices while retaining both analyte and matrix concentrate,and facilitating them to form homogeneous co-crystals on the centrally located polystyrene pattern.With the OISE plate,high quality and reproducible spectra could be obtained for low abundance peptides even in the presence of high salt concentrations?200 mM NH₄HCO₃,1 M NaCl,or 400 mM urea?.Using this strategy,a significant sensitivity enhancement was gained over traditional MALDI plate.The practical utility of this method was further demonstrated by the successful profiling of BSA digests and human serum.4.?-Glutamyltranspeptidase?GGT?plays an important role in cellular glutathione/cysteine homeostasis and is a potential tumor biomarker.Herein,we rationally design and evaluate a GGT-specific probe Aq-ECG for quantitative analysis of GGT activity by MALDI-MS.The Aq-ECG was facilely synthesized by combining glutathione?ECG?as a recognition unit with a UV-absorptive aromatic anthraquinone?Aq?through a thiol-ene click reaction.In the presence of GGT,the cleavage of?-glutamyl group in Aq-ECG probe lead to a decrease in content,which can be accurately measured by using a nonisotopic internal standard?Aq-ECA,has a methyl difference with Aq-ECG?.Both Aq-ECG and Aq-ECA show high ionization ability with high-quality signal spectra without matrix interference and in-source dissociation.Using Aq-ECG and Aq-ECA,the GGT amount in the range of 1?50 U/L can be quantitatively determined by MALDI-MS.Also,this technique was used for determining the IC50 value of the GGT inhibitor acivicin.Furthermore,a magnetic graphene oxide?MGO?material was prepared and used for selective capture of Aq-ECG and Aq-ECA from complex real samples.Therefore,quantitative MALDI-MS analysis of the GGT levels in human serum samples from both healthy and liver cancer patients,and the GGT expressions in tumor/normal cell lysate can be readily achieved by using Aq-ECG and Aq-ECA,in combination with an enrichment process by MGO.In general,the proposed probe system along with its enrichment strategy represents a simple and robust tool for rapid screening of GGT activity.