| Matrix-assisted laser desorption/ionization(MALDI)mass spectrometry has become an important tool in proteomics,metabolomics,glycomics,microbial and nucleic acid detection due to its high sensitivity,high throughput,high salt tolerance,simplicity and rapidity.However,direct analysis of some compounds that are difficult to ionize,strong polarity and unstable via MALDI MS is still difficult.Moreover,the traditional matrix could produce many fragments in low m/z region,which will interfere with the signal of the analyte.For some biomolecules,such as carbohydrates,they have low ionization efficiency because of their hydrophilic nature,low abundance and low proton affinity.Therefore,carbohydrates are necessary to be enriched and derived before MALDI MS analysis.Common sample pretreatment steps and derivatization methods are often followed by complex,tedious and timeconsuming steps,which lead to the loss of samples.Furthermore,the non-uniform crystallization of traditional matrix leads to poor reproducibility of MALDI MS,which is not suit for quantitative analysis.It is difficult to achieve the direct analysis of target compounds because the composition of biological samples is complex.Therefore,it makes sense to develop some derivatization reagents with matrix effects to selectively improve the ionization efficiency of target analytes,avoid complex sample processing steps,replace the use of traditional matrix,and directly detect target biomolecules from complex biological samples.the following three works have been carried out in this paper:The detection of carbohydrates by MALDI MS is often limited by the unsatisfactory ionization efficiency,instability and the matrix interference in low molecular weight region.Herein,a rational designed reactive matrix,2-hydrazinoquinoline(2-HQ),can be used to detect neutral,sialic and low molecular weight carbohydrates sensitively both in the positive and negative ion mode.Since 2-HQ reacts efficiently with the reducing end of carbohydrate to form stable hydrazone,the ionization efficiency of derived carbohydrates is significantly enhanced.Moreover,the quinoline structure of 2-HQ makes it have strong matrix effect.Therefore,excessive 2-HQ could be used as matrix directly without removing.Using 2-HQ as matrix,the sensitivity for analyzing glycans has been improved 10-fold and 100-fold compared with those using 3-aminquinoline(3-AQ)and 2,5-dihydroxybenzoic acid(DHB)as matrix,respectively.Moreover,quantitative analysis of neutral,acidic and low molecular weight carbohydrates has been achieved because of the good reproducibility by using 2-HQ as matrix.As a result,up to 50 glycans in a single sample spot of human fresh serum without any prior purification and enrichment have been successfully detected.Therefore,2-HQ as a new reactive matrix has shown great potentials in widespread applications for sensitive,selective,quantitative,high speed and high throughput analysis of carbohydrates in complex samples by MALDI-TOF MS.Human milk oligosaccharides(HMOs)play important biological roles in infant growth and development.Therefore,the analysis of HMOs is helpful to understand the relationship between their structure and function.However,the direct analysis of HMOs remains challenging because of the complex composition of human milk.In this paper,we applied 2-HQ to the analysis of human milk oligosaccharides due to its strong ability in the analysis of glycans in complex biological samples.After the removal of lactose,the HMOs was tagged directly on the MALDI target plate without the separation of the neutral and acidic oligosaccharides,which simplified the operation procedure and achieved the simple,rapid and sensitive analysis of free oligosaccharides in human milk.In addition,we compared the differences of oligosaccharides in human milk and animal milk,the result showed that animal milk contains only one or two kinds of oligosaccharides,which cannot replace the role of human milk in infant growth.Subsequently,we compared the differences of HMOs at different lactation stages,it turns out that the oligosaccharides decreased gradually with the increase of lactation,especially sialylated oligosaccharides and oligosaccharides in high m/z region.In recent years,MALDI MS is an effective tool for the analysis of enzyme activities,However,synthetic substrates had negative effects on enzyme activity.There are still some challenges in using natural substrates for analysis of enzyme activities due to the low ion intensity of natural substrates in MS.In this paper,we developed a simple,sensitive and quantitative method for the determination of sialidase activity.Past works showed that 2-HQ has a great advantage compared with other matrix and derivatization reagents in the detection of acidic carbohydrates,have no matrix interference in the low molecular region in negative ion mode.In this work,we used natural substrates and 2-HQ to complete the one-step identification and labeling of sialidase hydrolysates on the target plate and direct analysis in MALDITOF MS.In this work,natural carbohydrate 3’-sialyllactose(3’-SL)was used as enzyme substrate and products were directly identified and immobilized on the target plate after the enzyme-catalyzed reaction,then analyzed in negative ion mode without the additional matrix.The results showed that the signal intensity of acid substrate and product were significantly improved,and the signal intensity of neutral product was almost suppressed.There is no interference of cationic adduction peaks and matrix background.This method could be used for quantitative analysis of sialidase activity without the internal standard.In addition,this method has been successfully applied to the screening of inhibitors and analysis of substrate specificity. |
