ABSTRACT
Objective@#To optimize the sample pretreatment and establish an ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-MS) assay based on the parallel reaction monitoring (PRM) mode for determination of antibiotic residues in chicken meat.@*Methods@#Blank matrix-spiked chicken meat samples were extracted with 95% acetonitrile aqueous solution containing Na2EDTA and formic acid. The extraction solutions were cleaned up using different combinations of C18, PSA and GCB fillers, and the combinations with a higher antibiotic recovery rate was screened. The residues of 32 antibiotics were determined using UPLC-Q-Orbitrap-MS based on the PRM mode.@*Results@#If the extraction solution was cleaned up using the C18 filler, the largest number of antibiotics with a spiked recovery rate of >80% was seen, with matrix effects of 82.2% to 112.6%. The detection limits of 32 antibiotics were 0.8 to 5.8 μg/kg, with linear correlation coefficients of >0.99, spiked recovery rates of 71.3% to 111.5% and relative standard deviations of 3.2% to 14.2%.@*Conclusion@#The UPLC-Q-Orbitrap-MS assay is suitable for determination and quantitative analysis of multiple antibiotics in chicken meat. Key words： high-resolution mass spectrometry orbitrap antibiotic residue parallel reaction monitoring
ABSTRACT
Xiaoer-Feire-Kechuan (XFK) is an 11-herb Chinese medicine formula to treat cough and pulmonary inflammation.The complicated composition rendered its chemical analysis and effective-component elucidation.In this study,we combined quantitative analysis and bioactivity test to reveal the anti-inflammatory constituents of XFK.First,UPLC-DAD and UHPLC/Q-Orbitrap-MS methods were estab-lished and validated to quantify 35 analytes (covering 9 out of 11 herbs) in different XFK formulations.Parallel reaction monitoring mode built in Q-Orbitrap-MS was used to improve the sensitivity and selectivity.Then,anti-inflammatory activities of the 35 analytes were analyzed using in vitro COX-2 inhibition assay.Finally,major analytes forsythosides H,I,A (8-10),and baicalin (15) (total contents varied from 21.79 to 91.20 mg/dose in different formulations) with significant activities (inhibitory rate ≥ 80％) were proposed as the anti-inflammatory constituents of XFK.The present study provided an effective strategy to discover effective constituents of multi-herb formulas.
ABSTRACT
Magnolia officinalis is a traditional Chinese medicine,with many years of cultivating process, M. officinalis leaves show more differentiation types due to the exchange of seeds from different provenances. "Da Ao"(DA), "Xiao Ao"(XA), "Chuan Hou"(CH),and "Liu Ye"(LY)are the main types of M. officinalis in Sichuan province of China,and there were obvious differences in growth rate,chemical composition,leaf shape and leaf colour. This study selected different types of M. officinalis leaves(DA,XA,LY and CH)from Sichuan to determine their chlorophyll content. Transcriptomic level sequencing of different types of M. officinalis leaf tissues was by high-throughput sequencing analysis and proteomics used an integrated approach involving TMT labelling and LC-MS/MS to quantify the dynamic changes of the whole proteome of M. officinalis. The results showed that CH had the lowest chlorophyll content while DA had the highest chlorophyll content. Furthermore,transcriptome and proteomics results showed that chlorophyll synthesis pathway in DA glutamine-tRNA reductase,urinary porphyrins decarboxylase(UROD),oxygen-dependent protoporphyrin(ODCO),the original-Ⅲ oxidase protoporphyrin oxidase(PPO),magnesium chelating enzyme subunit ChlD,protoporphyrin magnesium Ⅸ monomethyl ester [oxidative] cyclase(MPPMC)were significantly higher than CH,XA and LY,consistent in the results of determination of chlorophyll content(chlorophyll content was highest of 37.56 mg·g~(-1) FW). Some rate-limiting enzymes related to the chlorophyll synthesis,such as ODCO,PPO and MPPMC were tested by Parallel Reaction Monitoring(PRM),and the results showed that the rate-limiting enzyme content in DA was higher than that in other three types. Therefore,based on the differences in leaf color of four types of M. officinalis,the research conducted a preliminary study on the chlorophyll metabolism pathway in leaves of different types of M. officinalis,and explored relevant genes and proteins causing leaf color differences from the molecular level,so as to lay a foundation for studying the differences in growth and development of different types of M. officinalis.
Subject(s)
China , Chlorophyll , Chromatography, Liquid , Magnolia , Plant Leaves , Proteome , Tandem Mass Spectrometry , TranscriptomeABSTRACT
Proteomics represented vital applications of technologies in the identification and quantification of high to moderate proteins (cellular signalling networks) found in biological matrix such as tissues, cells and fluids. Proteomics based technical knowledge is applied and verified in several preclinical research settings such as invention of diagnostic markers for specific disease and have shown to be increased in clinical applications. Extensive studies on proteomics resulted in detection of biomarkers that have been highly advanced in using diseases for cancer, lungs, cardiovascular, renal and neuro-regenerative and Parkinson's disease by introducing human origins for biocompatibility such as urine and serum. Advancement in the proteomic methods is conferring candidate right direction for clinical usage. In this review, recent developments and widely used proteomics approaches such as Mass Spectrometry (MS), Microarray chips are elaborately addressed and also focused merits and demerits of commonly used advanced approaches such as Selected Reaction Monitoring (SRM), Parallel Reaction Monitoring (PRM) and Data Independent Acquisition (DIA) and other used proteomics and that roles, in order to aid clinicians, were also discussed in the light of biomedical applications.
ABSTRACT
The bevacizumab and its glycoforms were analyzed by matrix-assisted laser desorption ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-MS), sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE) and short-gun strategy, with the sequence of unique peptide and seventeen glycoforms being characterized. The bevacizumab and its glycopeptides concentrations in mice plasma with different intravenous injection doses of bevacizumab were detected and the concentration-time curves were obtained by parallel reaction monitoring ( PRM) method based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technique. First, standard curves were created for quantification of mAb in mice plasma, which showed good linearity, with the correlation coefficient (R2 ) value of 0. 998 and the lower limit of quantification of 66 fmol. Detection results of high and low doses of the drug in the mice plasma samples showed that the drug concentration-time curve trend was consistent, e. g. the concentration was decreasing. However, the results of quantitation of seventeen glycoforms demonstrated that the metabolism of different glycoforms was different. The concentrations of most glycoforms increased first, whereas the metabolism afterwards differed by different glycoforms.
ABSTRACT
Steady improvement in mass spectrometers technology has transformed the targeted proteome analysis into a new stage. Parallel reaction monitoring (PRM) technology has evolved from the basic multiple reaction monitoring (MRM) targeted proteomics methods in recent years. PRM performs with a higher sensitivity, throughput and reproducibility in targeted quantification, however its limitations in effectiveness and accurate quantification of samples with higher complexity still remain unsolved. In this study through improving the chromatographic conditions of PRM we established a simple and robust platform for targeted proteomic quantification. The newly established PRM system is equipped with columns with increased inner diameter (150 μm) and decreased total length (8 cm); faster liquid phase elution rate (800 nL/min) and shortened elution gradient (35 min). These modifications enable PRM platform to combine with dual reverse phase chromatography, to quantify up to 400 low abundance peptides in human 293T cells whole cell extract. Our findings would benefit the promotion of PRM technology, especially providing a technical option for accurate quantification of low abundance proteins.
ABSTRACT
Mass spectrometry is an important and powerful tool for protein quantification. With the in-depth development of quantitative proteomics, limitations of classic MS based quantification methods, such as complicated matrix interference and throughput/capacity limitation, start to appear. Recent progress of series novel MS based techniques provide effective solutions for the limitations of relative and absolute proteomic quantification, including synchronous precursor selection ( SPS ) , mass defect isobaric labeling, parallel reaction monitoring ( PRM) , multiplexing acquisition ( MSX) , and various novel data independent acquisition ( DIA) modes. Here we summarized the current limitations of quantitative proteomics, reviewed the latest MS based quantification approaches, and discussed the features and advantages of these novel techniques for quantitative proteomic application.