scholarly journals A site-specific map of the human plasma glycome and its age and gender-associated alterations

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Alexander A. Merleev ◽  
Dayoung Park ◽  
Yixuan Xie ◽  
Muchena J. Kailemia ◽  
Gege Xu ◽  
...  
Keyword(s):  
Human Plasma ◽  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Spectrometry Method ◽  
Age And Gender ◽  
Site Specific ◽  
And Gender ◽  
Biomarker Research ◽  
A Site ◽  
Age Prediction

Abstract Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR < 0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r2 = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.

scholarly journals Development of UHPLC-MS/MS Method for Indirubin-3′-Oxime Derivative as a Novel FLT3 Inhibitor and Pharmacokinetic Study in Rats

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2039
Author(s):  
Na Yoon Kim ◽  
Yong-Chul Kim ◽  
Yoon Gyoon Kim
Keyword(s):  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Rat Plasma ◽  
Reaction Monitoring ◽  
Spectrometry Method ◽  
Limit Of Quantification ◽  
Flt3 Inhibitor ◽  
Pharmacokinetic Properties ◽  
Safety Guidelines ◽  
The U.S

This study aimed to develop and validate a sensitive liquid chromatography-coupled tandem mass spectrometry method for the quantification of LDD-2614, an indirubin derivative and novel FLT3 inhibitor, in rat plasma. In addition, the developed analytical method was applied to observe the pharmacokinetic properties of LDD-2614. Chromatographic separation was achieved on a Luna omega C18 column using a mixture of water and acetonitrile, both containing 0.1% formic acid. Quantitation was performed using positive electrospray ionization in a multiple reaction monitoring (MRM) mode. The MRM transitions were optimized as m/z 426.2→113.1 for LDD-2614 and m/z 390.2→113.1 for LDD-2633 (internal standard), and the lower limit of quantification (LLOQ) for LDD-2614 was determined as 0.1 ng/mL. Including the LLOQ, the nine-point calibration curve was linear with a correlation coefficient greater than 0.9991. Inter- and intraday accuracies (RE) ranged from −3.19% to 8.72%, and the precision was within 9.02%. All validation results (accuracy, precision, matrix effect, recovery, stability, and dilution integrity) met the acceptance criteria of the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety guidelines. The proposed method was validated and demonstrated to be suitable for the quantification of LDD-2614 for pharmacokinetics studies.

scholarly journals Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma

2009 ◽  
Vol 8 (8) ◽  
pp. 1860-1877 ◽  
Author(s):  
Michael A. Kuzyk ◽  
Derek Smith ◽  
Juncong Yang ◽  
Tyra J. Cross ◽  
Angela M. Jackson ◽  
...  
Keyword(s):  
Human Plasma ◽  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Absolute Quantitation

scholarly journals Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin

2021 ◽  
Vol 9 ◽  
Author(s):  
Ding Liu ◽  
Shuaishuai Wang ◽  
Junping Zhang ◽  
Weidong Xiao ◽  
Carol H. Miao ◽  
...  
Keyword(s):  
Human Plasma ◽  
Collision Energy ◽  
T Antigen ◽  
Plasma Fibronectin ◽  
Site Specific ◽  
Adhesive Protein ◽  
Human Fibronectin ◽  
A Site ◽  
Functional Analyses ◽  
Labeling Method

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.

scholarly journals Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple- Reaction–Monitoring Mass Spectrometry

2010 ◽  
Vol 56 (12) ◽  
pp. 1804-1813 ◽  
Author(s):  
Sean A Agger ◽  
Luke C Marney ◽  
Andrew N Hoofnagle
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
Apolipoprotein B ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Reaction Monitoring ◽  
Normal Flow ◽  
Apolipoprotein A ◽  
Deming Regression

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs &lt;6% and interassay CVs &lt;12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay − 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.

Liquid chromatography–tandem mass spectrometry method for the quantification of a potent H3 receptor antagonist conessine in serum and its application to pharmacokinetic studies

2018 ◽  
Vol 24 (3) ◽  
pp. 289-298
Author(s):  
Mahendra Shukla ◽  
Femi M Francis ◽  
Jawahar Lal
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Multiple Reaction Monitoring ◽  
Pharmacokinetic Studies ◽  
Tandem Mass ◽  
Reaction Monitoring ◽  
Spectrometry Method ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Conessine, a steroidal alkaloid obtained from the bark and seeds of the plant species of Apocynaceae family, elicits a histamine antagonistic action, selectively for the H3 histaminergic receptors. This alkaloid is used mainly for the treatment of dysentery and helminthic disorders. For the quantification of conessine in serum, a liquid chromatography–tandem mass spectrometry method was developed. Chromatographic separation was achieved on a Zorbax SB-CN column (100 × 4.6 mm, 3.5 µm), and a mobile phase consisting of 90% methanol in aqueous ammonium acetate buffer (pH 3.5) with 0.1% (v/v) formic acid at an isocratic flow rate of 0.6 ml/min at 40℃ provides efficiency in separation. A volume of 40 µl was injected each time and the run time for each sample was 5 min. Phenacetin (internal standard) was added to 50 µl of serum sample prior to liquid–liquid extraction using 3% isopropanol in n-hexane. The detection was performed on a 5500 QTRAP mass spectrometer by multiple reaction monitoring mode via electrospray ionization source. The multiple reaction monitoring of conessine and IS was m/ z 357.4 to m/ z 312.1 and m/ z 180.1 to m/ z 138.1, respectively. The method that showed selectivity and linearity in the range of 1–200 ng/ml was validated in terms of sensitivity, accuracy, precision and stability. The detection and quantitation limits were recognized at 0.1 and 1 ng/ml, respectively. The intra- and inter-day precision and accuracy fulfils the acceptance criteria. Applying the method to the pharmacokinetic studies in rats, conessine showed a peak serum concentration at 2 h post oral dose with a good bioavailability of 71.28 ± 4.65%.

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