scholarly journals Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria

2020 ◽  
Vol 21 (14) ◽  
pp. 4924 ◽  
Author(s):  
Anna A. Toymentseva ◽  
Anastasia O. Koryagina ◽  
Alexander V. Laikov ◽  
Margarita R. Sharipova
Keyword(s):  
Serine Proteases ◽  
Multiple Reaction Monitoring ◽  
Expression System ◽  
Complex Mixture ◽  
Rapid Identification ◽  
Glutamyl Endopeptidase ◽  
Reaction Monitoring ◽  
Label Free ◽  
Fragment Ions ◽  
Specific Proteins

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.

scholarly journals Label-Free Absolute Quantitation of Oligosaccharides Using Multiple Reaction Monitoring

2014 ◽  
Vol 86 (5) ◽  
pp. 2640-2647 ◽  
Author(s):  
Qiuting Hong ◽  
L. Renee Ruhaak ◽  
Sarah M. Totten ◽  
Jennifer T. Smilowitz ◽  
J. Bruce German ◽  
...  
Keyword(s):  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free ◽  
Absolute Quantitation

scholarly journals Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments

Biology ◽  
2014 ◽  
Vol 3 (2) ◽  
pp. 383-402 ◽  
Author(s):  
Lisa Chung ◽  
Christopher Colangelo ◽  
Hongyu Zhao
Keyword(s):  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free

scholarly journals Rapid Verification of Candidate Serological Biomarkers Using Gel-based, Label-free Multiple Reaction Monitoring

2011 ◽  
Vol 10 (9) ◽  
pp. 4005-4017 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Lynn A. Beer ◽  
Kurt T. Barnhart ◽  
David W. Speicher
Keyword(s):  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free ◽  
Serological Biomarkers

Quantitative analysis of genetically modified soya using multiple reaction monitoring mass spectrometry with endogenous peptides as internal standards

2018 ◽  
Vol 25 (1) ◽  
pp. 50-57 ◽  
Author(s):  
Shobha Devi ◽  
Yi-Cheng Lin ◽  
Yen-Peng Ho
Keyword(s):  
Mass Spectrometry ◽  
Genetically Modified ◽  
Multiple Reaction Monitoring ◽  
Linear Regression Analysis ◽  
Exchange Chromatography ◽  
Protein Quantification ◽  
Reaction Monitoring ◽  
Label Free ◽  
Internal Standards ◽  
Phosphate Synthase

A simple label-free method was developed for the quantification of the herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase using multiple reaction monitoring liquid chromatography–mass spectrometry. Sample pretreatment procedures including ion exchange chromatography and CaCl2 precipitation were used to purify the 5-enolpyruvylshikimate-3-phosphate synthase protein. Quantification of various percentages of genetically modified soya (0.5–100%) was performed by selecting suitable endogenous soybean peptides as internal standards. Results indicated that Gly P (QGDVFVVPR) and Lec P (LQLNK) are useful internal standards for the quantification of low and high percentages of genetically modified soya, respectively. Linear regression analysis of both calibration curves yielded good linearity with R2 of 0.99. This approach is a convenient and accurate quantification method for genetically modified soya at a level as low as 0.5% (less than the current EU threshold for labeling genetically modified soya).

scholarly journals Multiple-Reaction Monitoring–Mass Spectrometric Assays Can Accurately Measure the Relative Protein Abundance in Complex Mixtures

2012 ◽  
Vol 58 (4) ◽  
pp. 777-781 ◽  
Author(s):  
Andrew N Hoofnagle ◽  
Jessica O Becker ◽  
Michael N Oda ◽  
Giorgio Cavigiolio ◽  
Philip Mayer ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Dilution ◽  
Matrix Effects ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Shotgun Proteomics ◽  
Mass Spectrometric ◽  
Reaction Monitoring ◽  
Label Free ◽  
Operating Characteristics

Abstract BACKGROUND Mass spectrometric assays could potentially replace protein immunoassays in many applications. Previous studies have demonstrated the utility of liquid chromatography–multiple-reaction monitoring–mass spectrometry (LC-MRM/MS) for the quantification of proteins in biological samples, and many examples of the accuracy of these approaches to quantify supplemented analytes have been reported. However, a direct comparison of multiplexed assays that use LC-MRM/MS with established immunoassays to measure endogenous proteins has not been reported. METHODS We purified HDL from the plasma of 30 human donors and used label-free shotgun proteomics approaches to analyze each sample. We then developed 2 different isotope-dilution LC-MRM/MS 6-plex assays (for apoliporoteins A-I, C-II, C-III, E, B, and J): 1 assay used stable isotope-labeled peptides and the other used stable isotope-labeled apolipoprotein A-I (an abundant HDL protein) as an internal standard to control for matrix effects and mass spectrometer performance. The shotgun and LC-MRM/MS assays were then compared with commercially available immunoassays for each of the 6 analytes. RESULTS Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. In contrast, the isotope dilution LC-MRM/MS approaches showed correlations with immunoassays of r = 0.61–0.96. The LC-MRM/MS approaches had acceptable reproducibility (<13% CV) and linearity (r ≥0.99). Strikingly, a single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards. CONCLUSIONS Because peak area ratios measured in multiplexed LC-MRM/MS assays correlate well with immunochemical measurements and have acceptable operating characteristics, we propose that LC-MRM/MS could be used to replace immunoassays in a variety of settings.

Tandem Mass Spectrometry in Physiology

Physiology ◽  
2007 ◽  
Vol 22 (6) ◽  
pp. 390-400 ◽  
Author(s):  
Trairak Pisitkun ◽  
Jason D. Hoffert ◽  
Ming-Jiun Yu ◽  
Mark A. Knepper
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Recent Progress ◽  
Multiple Reaction Monitoring ◽  
Complex Mixture ◽  
Shotgun Proteomics ◽  
Cell Physiology ◽  
Tandem Mass ◽  
Reaction Monitoring

Tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) allows identification of proteins in a complex mixture without need for protein purification (“shotgun” proteomics). Recent progress in LC-MS/MS-based quantification, phosphoproteomic analysis, and targeted LC-MS/MS using multiple reaction monitoring (MRM) has made LC-MS/MS a powerful tool for the study of cell physiology.

scholarly journals Quantification of Intact O-Glycopeptides on Haptoglobin in Sera of Patients With Hepatocellular Carcinoma and Liver Cirrhosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Hong Shu ◽  
Lei Zhang ◽  
Yiwei Chen ◽  
Yijie Guo ◽  
Limin Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Liver Cirrhosis ◽  
Acute Phase Response ◽  
Lectin Binding ◽  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Label Free ◽  
Comprehensive Understanding ◽  
Lectin Blot ◽  
Free Quantification

Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC. Furthermore, label-free quantification based on MS was performed. A total of 26 intact O-glycopeptides were identified on Hp, and most of them were elevated in HCC as compared to LC. Among them, the intensity of HYEGS316TVPEK (H1N1S1) on Hp was the highest in HCC patients. Increased HYEGS316TVPEK (H1N1S1) in HCC was quantified and confirmed using the MS method based on 18O/16O C-terminal labeling and multiple reaction monitoring. This study provided a comprehensive understanding of the glycosylation of Hp in liver diseases.

scholarly journals EISA-MRM: Multiple Reaction Monitoring with Single Quadrupole Mass Spectrometry

2021 ◽  
Author(s):  
Jingchuan Xue ◽  
Rico J.E. Derks ◽  
William Wwebb ◽  
Aries Aisporna ◽  
Martin Giera ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Analysis ◽  
Dynamic Range ◽  
Matrix Effects ◽  
Multiple Reaction Monitoring ◽  
Mass Spectrometry Analysis ◽  
Reaction Monitoring ◽  
Quadrupole Mass ◽  
Fragment Ions ◽  
Specificity And Sensitivity

<p>Enhanced in-source fragmentation annotation combined with single quadrupole multiple reaction monitoring (EISA-MRM) has been designed for quantitative mass spectrometry analysis. EISA contrasts to traditional electrospray as a soft ionization technology and is proving to be advantageous since the resulting fragment ions are identical as those generated in tandem mass spectrometry. Criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis was used for the EISA-MRM quantitative analyses and experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for quantitative analysis was comparable to QqQ-MRM analyses at up to 5 orders of magnitude and the EISA-MRM and QqQ-MRM of the cell and plasma extracts showed similar matrix effects. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labelled standards demonstrated EISA-MRM accuracy in the range of 91-110% for the amino acids, 76-129% for the fatty acids, and precision with a CV < 10%. In order to enhance specificity and sensitivity, a newly developed Correlated SIM Chromatogram (CSC) algorithm was designed to facilitate MRM quality analyses. The EISA-MRM quantitative analysis with CSC informatics enables both precursor and in-source fragment ions to be correlated within a single quadrupole mass spectrometer.</p>

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