Circulating choline and phosphocholine measurement by a hydrophilic interaction liquid chromatography–tandem mass spectrometry
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ABSTRACT:
SUBMITTER: Guerra G
PROVIDER: S-EPMC10665723 | biostudies-literature | 2023 Nov
REPOSITORIES: biostudies-literature
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Project description:Protein phosphorylation is a critical post-translational modification (PTM). Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides with enriched regions of serines, threonines, and tyrosines that often orchestrate critical biological functions. To address this issue, we developed a simple, easily implemented method to introduce a commonly used tandem mass tag (TMT) to increase peptide hydrophobicity, effectively enhancing RPLC-MS analysis of hydrophilic peptides. Different from conventional TMT labeling, this method capitalizes on using a nonprimary amine buffer and TMT labeling occurring before C18-based solid phase extraction. Through phosphoproteomic analyses of MCF7 cells, we have demonstrated that this method can greatly increase the number of identified hydrophilic phosphopeptides and improve MS detection signals. We applied this method to study the peptide QPSSSR, a very hydrophilic tryptic peptide located on the C-terminus of the G protein-coupled receptor (GPCR) CXCR3. Identification of QPSSSR has never been reported, and we were unable to detect it by traditional methods. We validated our TMT labeling strategy by comparative RPLC-MS analyses of both a hydrophilic QPSSSR peptide library as well as common phosphopeptides. We further confirmed the utility of this method by quantifying QPSSSR phosphorylation abundances in HEK 293 cells under different treatment conditions predicted to alter QPSSSR phosphorylation. We anticipate that this simple TMT labeling method can be broadly used not only for decoding GPCR phosphoproteome but also for effective RPLC-MS analysis of other highly hydrophilic analytes.
| S-EPMC7197904 | biostudies-literature
Project description:BackgroundThe measurement of plasma concentrations of retinol binding protein is a component of nutritional assessment in neonatal intensive care. However, serial testing in newborns is hampered by the limited amount of blood that can be sampled. Limitations are most severe with preterm infants, for whom close monitoring may be most important.MethodsWe developed an assay to quantify retinol binding protein using trypsin digestion and liquid chromatography-tandem mass spectrometry, which requires a serum or plasma volume of 5 µl. Additionally, we validated the method according to current recommendations and performed comparison with a standard nephelometry platform in clinical use.ResultsThe assay demonstrated linearity from below 1 mg/dL (0.48 µM) to more than 20 mg/dL (9.7 µM), and an imprecision of 11.8% at 0.43 mg/dL (0.21 µM). The distribution of results observed with the new method was different when compared with nephelometry.ConclusionLiquid chromatography-tandem mass spectrometry facilitated testing a smaller sample volume, thereby increasing the ability to monitor key nutritional markers in premature infants. The differences in results compared with a commercially-available nephelometric assay revealed questionable results for lower concentrations by immunoassay.
| S-EPMC8671195 | biostudies-literature
Project description:Background. The clinical relevance of circulating 1,25-dihydroxyvitamin D (1,25(OH)2D) is probably underappreciated, but variations in the measurement of this difficult analyte between different methods limit comparison of results. Methods. In 129 clinical samples, we compared a new automated assay with a commercially available liquid chromatography tandem mass spectrometry (LC-MS/MS) kit. Results. Median (interquartile range) 1,25(OH)2D concentrations with the automated assay and the LC-MS/MS method were 26.6 pg/mL (18.5-39.0 pg/mL) and 23.6 pg/mL (16.1-31.3 pg/mL), respectively (P = 0.001). Using the method-specific cut-offs for deficient 1,25(OH)2D levels (<20 pg/mL for the automated assay and <17 pg/mL for the LC-MS/MS method), the percentage of patients classified as 1,25(OH)2D deficient was 28.7% and 27.1%, respectively. However, concordance between the two methods for deficient levels was only 62% and the concordance correlation coefficient was poor (0.534). The regression equation resulted in an intercept of -1.99 (95% CI: -7.33-1.31) and a slope of 1.27 (95% CI: 1.04-1.52) for the automated assay. The mean bias with respect to the mean of the two methods was -3.8 (1.96 SD: -28.3-20.8) pg/mL for the LC-MS/MS method minus the automated assay. Conclusions. The two methods show only modest correlation and further standardization is required to improve reliability and comparability of 1,25(OH)2D test procedures.
| S-EPMC4835639 | biostudies-literature
Project description:The proliferation of cyanobacteria can result in algal blooms, which may cause environmental and biological harm due to the production and release of secondary metabolites, or cyanotoxins, into the affected waterway. Cyanobacteria can produce multiple classes of cyanotoxins; therefore, to understand the full toxic load of algal blooms, it is necessary to perform analyses that quantify each class. These classes are generally monitored individually due to the challenges associated with the differing physicochemical properties of the cyanotoxins. Hydrophilic interaction liquid chromatography (HILIC) is a form of chromatography capable of retaining multiple classes of cyanotoxins that differ in physicochemical properties. Here an HILIC-MS/MS method was developed and validated to detect 3 microcystins, 11 saxitoxins, and 2 anatoxins. The chromatographic conditions were optimized to allow for the separation of multiple pairs of saxitoxin epimers, and in-source fragmentation in the MS interface was used to develop unique MRMs between the pairs. The method was validated and had low limits of detection (LODs, between 0.00770 and 9.75 µg L-1) and limits of quantification (LOQs, between 0.0257 and 32.5 µg L-1) for all compounds. All analytes exhibited good linearity (R2 values ≥ 0.991) and low percentage relative standard deviations for retention time (0%-1.74%) and peak area (4.54%-27.6%), with spiked recoveries ranging from 75.6% to 117% for all compounds. A multiclass sample preparation method to extract the three classes of analytes from cyanobacterial samples was developed and validated, with 80:20 acetonitrile:water and 0.1% formic acid as the optimal extraction solvent. The newly developed sample preparation and analysis methods were applied to cultured cyanobacteria and field samples, with microcystins and saxitoxins detected. The multiclass sample preparation and analysis methods developed here improve on individual methods as they reduce the complexity and time of sample preparation and analysis and will assist ecotoxicologists in assessing the full toxic risk of cyanobacterial blooms.
| S-EPMC11923511 | biostudies-literature
Project description:Mass spectrometry is a powerful analytical tool used for the analysis of a wide range of substances and matrices; it is increasingly utilized for clinical applications in laboratory medicine. This Primer includes an overview of basic mass spectrometry concepts, focusing primarily on tandem mass spectrometry. We discuss experimental considerations and quality management, and provide an overview of some key applications in the clinic. Lastly, the Primer discusses significant challenges for implementation of mass spectrometry in clinical laboratories and provides an outlook of where there are emerging clinical applications for this technology. Tandem mass spectrometry is increasingly utilized for clinical applications in laboratory medicine. In this Primer, Thomas et al. discuss experimental considerations and quality management for implementing clinical tandem mass spectrometry in the clinic with an overview of some key applications.
| S-EPMC9735147 | biostudies-literature
Project description:IntroductionOcrelizumab is a monoclonal anti-CD20 antibody approved for the treatment of multiple sclerosis (MS). The clinical value of therapeutic drug monitoring (TDM) for this antibody in treatment of MS is unknown, and an adequately specific and precise quantitation method for ocrelizumab in patient serum could facilitate investigation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitation methods have been shown to have higher analytic specificity and precision than enzyme-linked immunosorbent assays.ObjectivesTo establish and validate an LC-MS/MS-based quantitation method for ocrelizumab.MethodsWe present an LC-MS/MS-based quantitation method using immunocapture purification followed by trypsinization and analysis by a triple quadrupole mass analyzer obtaining results within the same day.ResultsWe found that the ocrelizumab peptide GLEWVGAIYPGNGDTSYNQK (Q1/Q3 Quantifier ion: 723.683+/590.77 y112+ Qualifier ion: 723.683+/672.30 y122+) can be used for quantitation and thereby developed a method for quantifying ocrelizumab in human serum with a quantitation range of 1.56 to 200 µg/mL. The method was validated in accordance with EMA requirements in terms of selectivity, carry-over, lower limit of quantitation, calibration curve, accuracy, precision and matrix effect. Ocrelizumab serum concentrations were measured in three MS patients treated with ocrelizumab, immediately before and after ocrelizumab infusion, with additional sampling after 2, 4, 8 and 12 weeks. Measured serum concentrations of ocrelizumab showed expected values for both Cmax and drug half-life over the sampled time period.ConclusionWe have established a reliable quantitation method for serum ocrelizumab that can be applied in clinical studies, facilitating the evaluation of ocrelizumab TDM in MS.
| S-EPMC9334332 | biostudies-literature
Project description:BackgroundMass spectrometry methods exhibit higher accuracy and lower variability than immunoassays at low testosterone concentrations. We developed and validated an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for quantifying serum total testosterone.MethodsWe used an ExionLC UPLC (Sciex, Framingham, MA, USA) system and a Sciex Triple Quad 6500+ (Sciex) MS/MS system in electrospray ionization and positive ion modes with multiple reaction monitoring transitions to evaluate precision, accuracy, linearity, lower limit of quantitation (LLOQ), carryover, ion suppression, stability, and reference intervals. For method comparison, we measured serum testosterone concentrations using this method in 40 subjects whose testosterone concentrations ranged from 0.14 to 55.48 nmol/L as determined using the Architect i2000 immunoassay (Abbott Diagnostics, Abbott Park, IL, USA) and in an additional 160 sera with testosterone concentrations <1.67 nmol/L.ResultsThe intra- and inter-run precision CVs were <2.81%, and the accuracy bias values were <3.85%, which were all acceptable. The verified linear interval was 0.03-180.84 nmol/L; the LLOQ was 0.03 nmol/L. No significant carryover and ion suppression were observed. The testosterone in serum was stable at 4°C, at -20°C, and after three freeze-thaw cycles. The reference intervals were successfully verified. The correlation was good at testosterone concentrations of 0.14-55.48 nmol/L; however, the Architect assay showed positive percent bias at concentrations <1.67 nmol/L.ConclusionsThe UPLC-MS/MS assay shows acceptable performance, with a lower LLOQ than the immunoassay. This method will enable the quantitation of low testosterone concentrations.
| S-EPMC9467846 | biostudies-literature
Project description:PurposeThe detection of hydrolysis products of Novichok agents in biological samples from victims is important for confirming exposure to these agents. However, Novichok agents are new class of nerve agent and there have been only few reports on analyses of Novichok agent degradation products. Here, we developed hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry (MS/MS) methods to detect Novichok agent degradation products in human urine with simple pretreatment and high sensitivity.MethodsA Poroshell 120 HILIC-Z column was used to analyze six Novichok agent degradation products. For urine samples, we used a simple pretreatment method, which consisted of deproteinization with acetonitrile and microfiltration. We calculated the pKa values of the OH groups, the log P values, and the molecular weights to investigate the difference in chromatographic behaviors of the Novichok agent degradation products and the degradation products of conventional nerve agents.ResultsSix Novichok agent degradation products, including N-(bis-(diethylamino)methylidene)-methylphosphonamidic acid (MPGA), which could not be detected by our previous method, could be analyzed with sufficient peak shape and mutual separation. The detection limits of six Novichok agent degradation products were sufficiently low (1-50 ng/mL) and the calibration curves showed sufficient linearity. The physicochemical parameters of Novichok agent degradation products were different from those of conventional nerve agent degradation products, and this explains the difference in chromatographic behaviors.ConclusionSix Novichok agent degradation products were successfully analyzed by HILIC-MS/MS. Due to the absence of a derivatization step, throughput performance was higher than our previous derivatization-liquid chromatography-MS/MS method.
| S-EPMC10310577 | biostudies-literature
Project description:Kefir, a fermented milk product produced using kefir grains, is a symbiotic consortium of bacteria and yeasts responsible for driving the fermentation process. In this study, an in-depth analysis of kefir's lipid profile was conducted, with a focus on its phospholipid (PL) content, employing liquid chromatography with high-resolution mass spectrometry (LC-HRMS). Nearly 300 distinct polar lipids were identified through hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization (ESI) and Fourier-transform orbital-trap MS and linear ion-trap tandem MS/MS. The identified lipids included phosphatidylcholines (PCs), lyso-phosphatidylcholines (LPCs), phosphatidylethanolamines (PEs) and lyso-phosphatidylethanolamines (LPEs), phosphatidylserines (PSs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The presence of lysyl-phosphatidylglycerols (LyPGs) was identified as a key finding, marking a lipid class characteristic of Gram-positive bacterial membranes. This discovery highlights the role of viable bacteria in kefir and underscores its probiotic potential. The structural details of minor glycolipids (GLs) and glycosphingolipids (GSLs) were further elucidated, enriching the understanding of kefir's lipid complexity. Fatty acyl (FA) composition was characterized using reversed-phase LC coupled with tandem MS. A mild epoxidation reaction with meta-chloroperoxybenzoic acid (m-CPBA) was performed to pinpoint double-bond positions in FAs. The dominant fatty acids were identified as C18:3, C18:2, C18:1, C18:0 (stearic acid), C16:0 (palmitic acid), and significant levels of C14:0 (myristic acid). Additionally, two isomers of FA 18:1 were distinguished: ∆9-cis (oleic acid) and ∆11-trans (vaccenic acid). These isomers were identified using diagnostic ion pairs, retention times, and accurate m/z values. This study provides an unprecedented level of detail on the lipid profile of kefir, shedding light on its complex composition and potential nutritional benefits.
| S-EPMC11818909 | biostudies-literature
Project description:Testosterone is a critical hormone involved in regulating various physiological processes in both men and women. Accurate testosterone measurement is essential for diagnosing endocrine disorders such as hypogonadism and polycystic ovary syndrome and for routine testing. Traditionally, testosterone levels are measured using serum or plasma samples, which present challenges in sample collection, storage, and transport, particularly in resource-limited settings. Dried blood spot (DBS) sampling has emerged as an effective alternative for hormone analysis, offering significant advantages in terms of sample stability, ease of collection, and simplified logistics. This study aimed to validate a DBS-based testosterone assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to ensure accuracy and precision comparable to conventional serum-based methods. Drops of whole blood samples were collected from adult volunteers using a single-use safety lancet for finger pricks, with blood applied onto DBS cards (PerkinElmer 226 Spot Saver RUO card) for further analysis. The testosterone was extracted from DBS using a liquid-liquid method and analyzed with LC-MS/MS. The assay demonstrated excellent linearity across a wide concentration range (0.1-100 ng/mL) with a correlation coefficient (r2) of 0.999 and achieved a lower limit of detection of 0.058 ng/mL and a lower limit of quantification of 0.086 ng/mL. The method showed high precision, with intra- and inter-day coefficients of variation below 10%, and satisfactory recovery rates. Hematocrit correction and matrix effect evaluations confirmed the robustness of the assay for clinical and research applications. Additionally, the assay displayed a strong clinical correlation between testosterone levels in DBS and venous serum samples, supporting its reliability for testosterone monitoring. This validation study supports that the DBS-based LC-MS/MS testosterone assay is a reliable tool for testosterone quantification for routine testing.
| S-EPMC11627179 | biostudies-literature