Recent progress in polar metabolite quantification in plants using liquid chromatography-mass spectrometry

Metabolite analysis or metabolomics is an important component of systems biology in the post-genomic era.Although separate liquid chromatography（LC） methods for quantification of the major classes of polar metabolites of plants have been available for decades,a single method that enables simultaneous determination of hundreds of polar metabolites is possible only with gas chromatography-mass spectrometry（GC-MS） techniques.The rapid expansion of new LC stationary phases in the market and the ready access of mass spectrometry in many laboratories provides an excellent opportunity for developing LC-MS based methods for multitarget quantification of polar metabolites.Although various LC-MS methods have been developed over the last 10 years with the aim to quantify one or more classes of polar compounds in different matrices,currently there is no consensus LC-MS method that is widely used in plant metabolomics studies.The most promising methods applicable to plant metabolite analysis will be reviewed in this paper and the major problems encountered highlighted.The aim of this review is to provide plant scientists,with limited to moderate experience in analytical chemistry,with up-to-date and simplified information regarding the current status of polar metabolite analysis using LC-MS techniques.

Inherently hydrophilic mesoporous channel coupled with metal oxide for fishing endogenous salivary glycopeptides and phosphopeptides

Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes.For in-depth profiling of glycosylation and phosphorylation,a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels(denoted as Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG).Based on the mechanism of hydrophilic interaction liquid chromatography(HILIC)and metal oxide affinity chromatography(MOAC),the Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides.With human saliva collected in successive four days as practical biological sample,endogenous glycopeptides and phosphopeptides are efficiently enriched.Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes.This strategy is anticipated to promote variation analysis of salivary post-translational modifications.

Determination of L-ergothioneine in food by UPLC-MS/MS method

L-Ergothioneine(L-EGT)possesses excellent antioxidant activity and has been used in the food,pharmaceuticals and cosmetics industries.In this study,a new efficient and sensitive ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method was established for the quantitative determination of L-EGT in food.The sample was extracted with methanol-water(70:30,V/V),separated by hydrophilic interaction liquid chromatography(HILIC)and detected by triple-quadrupole mass spectrometry.Validation studies were carried out on different product and the limit of quantitation was 20μg/kg(milk,alcohol-free beverages,dairy products)and 40µg/kg(cereal bars,chocolate).Excellent linearity(correlation coefficient(R2)≥0.999)was achieved for L-EGT quantification in the range of 5–200 ng/mL.The recoveries of the method(83.7%−107.5%)and the relative standard deviation(RSD,0.88%−6.84%(n=6))meet the performance criteria required for the determination of L-EGT in food.Finally,the applicability of the method was tested by analysing actual samples.In general,the method developed is simple,reliable,accurate,and stable and could be useful for routine analyses of L-EGT in food.

Evaluation and optimization of analytical procedure and sample preparation for polar Streptomyces albus J1074 metabolome profiling

Metabolomics is an essential discipline in omics technology that promotes research on the biology of microbial systems.Streptomyces albus J1074 is a model organism used in fundamental research and industrial microbiology.Nevertheless,a comprehensive and standardized method for analyzing the metabolome of S.albus J1074 is yet to be developed.Thus,we comprehensively evaluated and optimized the analytical procedure and sample preparation for profiling polar metabolites using hydrophilic interaction liquid chromatography(HILIC)coupled with high-resolution mass spectrometry(HRMS).We systematically examined the HILIC columns,quenching solutions,sample-to-quenching ratios,and extraction methods.Then,the optimal protocol was used to investigate the dynamic intracellular polar metabolite profile of the engineered S.albus J1074 strains during spinosad(spinosyn A and spinosyn D)fermentation.A total of 3648 compounds were detected,and 83 metabolites were matched to the standards.The intracellular metabolomic profiles of engineered S.albus J1074 strains(ADE-AP and OE3)were detected;furthermore,their metabolomes in different stages were analyzed to reveal the reasons for their differences in their spinosad production,as well as the current metabolic limitation of heterologous spinosad production in S.albus J1074.The HILIC-HRMS method is a valuable tool for investigating polar metabolomes,and provides a reference methodology to study other Streptomyces metabolomes.