Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory

Journal Name	Impact Factor	CiteScore	Launched Year	First Decision (median)	APC
Analytica analytica	-	1.8	2020	17.9 Days	CHF 1000	Submit
Journal of Clinical Medicine jcm	3.0	5.7	2012	16 Days	CHF 2600	Submit
Separations separations	2.5	3.0	2014	15.1 Days	CHF 2600	Submit
Biomolecules biomolecules	4.8	9.4	2011	18.4 Days	CHF 2700	Submit
Molecules molecules	4.2	7.4	1996	15.1 Days	CHF 2700	Submit
International Journal of Molecular Sciences ijms	4.9	8.1	2000	16.8 Days	CHF 2900	Submit

13 pages, 3040 KiB

Open AccessArticle

Development and Application of a UPLC–MRM–MS Method for Quantifying Trimethylamine, Trimethylamine-N-Oxide, and Related Metabolites in Individuals with and Without Metabolic Syndrome

by Mohammed E. Hefni and Cornelia M. Witthöft

Separations 2025, 12(2), 53; https://doi.org/10.3390/separations12020053 - 18 Feb 2025

Viewed by 472

Abstract

Trimethylamine-N-oxide (TMAO) is associated with various chronic diseases. TMAO is a downstream oxidative metabolite of trimethylamine (TMA) that is generated by the gut microbiota from dietary choline, carnitine, and betaine. Current analytical methods predominantly target TMAO only, due to the challenge of efficiently [...] Read more.

Trimethylamine-N-oxide (TMAO) is associated with various chronic diseases. TMAO is a downstream oxidative metabolite of trimethylamine (TMA) that is generated by the gut microbiota from dietary choline, carnitine, and betaine. Current analytical methods predominantly target TMAO only, due to the challenge of efficiently extracting and quantifying TMA. The present study demonstrates a simple and rapid UPLC–MRM–MS method for concurrent quantification of TMAO, TMA, and related precursors (choline, betaine, and various carnitines) following a methanol extraction from plasma and derivatization using iodoacetonitrile (IACN). Pure methanol resulted in a higher extractability of TMA (up to two-fold) compared to both pure acetonitrile and various methanol/acetonitrile mixtures. The quantification method showed high linearity within the tested range of 0.0625–100 μmol/L (determination coefficient > 0.999) and an intra- (n = 3) and inter-day (n = 9) precision of 2–8% along with an average recovery of above 94% for all metabolites (TMAO, TMA, choline, betaine, L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine). The method’s applicability was confirmed through a comparison of TMAO and its precursor concentrations in plasma samples of overnight-fasted subjects with (n = 12) and without (n = 21) metabolic syndrome. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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17 pages, 1798 KiB

Open AccessReview

Current Role and Potential of Triple Quadrupole Mass Spectrometry in Biomedical Research and Clinical Applications

by Andreas Tsakalof, Alexey A. Sysoev, Kira V. Vyatkina, Alexander A. Eganov, Nikolay N. Eroshchenko, Alexey N. Kiryushin, Alexey Yu. Adamov, Elena Yu. Danilova and Alexander E. Nosyrev

Molecules 2024, 29(23), 5808; https://doi.org/10.3390/molecules29235808 - 9 Dec 2024

Viewed by 1975

Abstract

Mass-spectrometry-based assays nowadays play an essential role in biomedical research and clinical applications. There are different types of commercial mass spectrometers on the market today, and triple quadrupole (QqQ) is one of the time-honored systems. Here, we overview the main areas of QqQ [...] Read more.

Mass-spectrometry-based assays nowadays play an essential role in biomedical research and clinical applications. There are different types of commercial mass spectrometers on the market today, and triple quadrupole (QqQ) is one of the time-honored systems. Here, we overview the main areas of QqQ applications in biomedicine and assess the current level, evolution, and trends in the use of QqQ in these areas. Relevant data were extracted from the Scopus database using the specified terms and Boolean operators defined for each field of the QqQ application. We also discuss the recent advances in QqQ and QqQ-based analytical platforms, which promote the clinical application of these systems, and explain the indicated substantial increase in triple quadrupole use in biomedicine. The number of biomedical studies utilizing QqQ increased 2–3 times this decade. Triple quadrupole is most intensively used in the field of endocrine research and testing. On the contrary, the relative rate of immunoassay utilization—a major competitor of chromatography–mass spectrometry—decreased in this area as well as its use within Therapeutic drug monitoring (TDM) and forensic toxicology. Nowadays, the applications of high-resolution accurate mass (HRAM) mass spectrometers in the investigated areas represent only a small fraction of the total amount of research using mass spectrometry; however, their application substantially increased during the last decade in the untargeted search for new biomarkers. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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31 pages, 6080 KiB

Open AccessArticle

Application of Human Plasma Targeted Lipidomics and Analysis of Toxic Elements to Capture the Metabolic Complexities of Hypothyroidism

by Anna Błażewicz, Michał Kiełbus, Katarzyna Skórzyńska-Dziduszko, Andreas M. Grabrucker, Jacqueline Jonklaas, Piotr Sosnowski, Alicja Trzpil, Anna Kozub-Pędrak, Agnieszka Szmagara, Julia Wojnicka, Ewelina Grywalska and Agostinho Almeida

Molecules 2024, 29(21), 5169; https://doi.org/10.3390/molecules29215169 - 31 Oct 2024

Cited by 1 | Viewed by 1186

Abstract

Background: Hypothyroidism (HT) affects millions worldwide and can lead to various lipid disorders. The metabolic complexity and the influence of toxic elements in autoimmune and non-autoimmune HT subtypes are not fully understood. This study aimed to investigate the relationships between plasma lipidome, toxic [...] Read more.

Background: Hypothyroidism (HT) affects millions worldwide and can lead to various lipid disorders. The metabolic complexity and the influence of toxic elements in autoimmune and non-autoimmune HT subtypes are not fully understood. This study aimed to investigate the relationships between plasma lipidome, toxic elements, and clinical classifications of HT in unexposed individuals. Methods: Samples were collected from 120 adults assigned to a study group with Hashimoto’s disease and non-autoimmune HT, and a healthy control group. Quantification of 145 pre-defined lipids was performed by using triple quadrupole tandem mass spectrometry (TQ MS/MS) in multiple reactions monitoring (MRM) mode via positive electrospray ionization (ESI). Levels of toxic elements were determined using inductively coupled plasma mass spectrometry (ICP-MS). Results: Significant associations between altered levels of several components of the plasma lipidome and Al, Cd, Ni, As, and Pb with HT were found. We show metabolic differences in lysophosphatidylcholines (LPC) and phosphatidylcholines (PC) between HT and controls, with distinct predicted activation patterns for lysolecithin acyltransferase and phospholipase A2. Conclusions: There are significant changes in the lipidome profiles of healthy subjects compared to euthyroid HT patients treated with L-thyroxine, which are related to the type of hypothyroidism and non-occupational exposure to toxic elements. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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15 pages, 4828 KiB

Open AccessArticle

An Integrated Approach Based on Clinical Data Combined with Metabolites and Biomarkers for the Assessment of Post-Operative Complications after Cardiac Surgery

by Peter Meinarovich, Alisa Pautova, Evgenii Zuev, Ekaterina Sorokina, Ekaterina Chernevskaya and Natalia Beloborodova

J. Clin. Med. 2024, 13(17), 5054; https://doi.org/10.3390/jcm13175054 - 26 Aug 2024

Cited by 1 | Viewed by 1204

Abstract

Background: Early diagnosis of post-operative complications is an urgent task, allowing timely prescribing of appropriate therapy and reducing the cost of patient treatment. The purpose of this study was to determine whether an integrated approach based on clinical data, along with metabolites and [...] Read more.

Background: Early diagnosis of post-operative complications is an urgent task, allowing timely prescribing of appropriate therapy and reducing the cost of patient treatment. The purpose of this study was to determine whether an integrated approach based on clinical data, along with metabolites and biomarkers, had greater predictive value than the models built on fewer data in the early diagnosis of post-operative complications after cardiac surgery. Methods: The study included patients (n = 62) admitted for planned cardiac surgery (coronary artery bypass grafting with cardiopulmonary bypass) with (n = 26) or without (n = 36) post-operative complications. Clinical and laboratory data on the first day after surgery were analyzed. Additionally, patients’ blood samples were collected before and on the first day after surgery to determine biomarkers and metabolites. Results: Multivariate PLS-DA models, predicting the presence or absence of post-operative complications, were built using clinical data, concentrations of metabolites and biomarkers, and the entire data set (ROC-AUC = 0.80, 0.71, and 0.85, respectively). For comparison, we built univariate models using the EuroScore2 and SOFA scales, concentrations of lactate, the dynamic changes of 4-hydroxyphenyllactic acid, and the sum of three sepsis-associated metabolites (ROC-AUC = 0.54, 0.79, 0.62, 0.58, and 0.70, respectively). Conclusions: The proposed complex model using the entire dataset had the best characteristics, which confirms the expediency of searching for new predictive models based on a variety of factors. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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16 pages, 4984 KiB

Open AccessArticle

Comparative Analysis of Serum Amino Acid Profiles in Patients with Myasthenia Gravis and Multiple Sclerosis

by Piotr Kośliński, Łukasz Rzepiński, Marcin Koba, Zdzisław Maciejek, Mariusz Kowalewski and Emilia Daghir-Wojtkowiak

J. Clin. Med. 2024, 13(14), 4083; https://doi.org/10.3390/jcm13144083 - 12 Jul 2024

Cited by 1 | Viewed by 1485

Abstract

Background: Multiple sclerosis (MS) and myasthenia gravis (MG) are autoimmune diseases that attack the central nervous system (CNS) and the neuromuscular junction, respectively. As the common pathogenesis of both diseases is associated with an autoimmune background and the involvement of T and [...] Read more.

Background: Multiple sclerosis (MS) and myasthenia gravis (MG) are autoimmune diseases that attack the central nervous system (CNS) and the neuromuscular junction, respectively. As the common pathogenesis of both diseases is associated with an autoimmune background and the involvement of T and B lymphocytes, the overlapping of selected clinical symptoms may cause difficulties in the differential diagnosis of both diseases. Methods: The aim of the study was to use Liquid Chromatography–Electrospray Ionization–Mass Spectrometry (LC–ESI–MS/MS) in conjunction with multivariate statistical analyses to examine the changes in amino acid metabolic profiles between patients with MG, MS, and a control group. Results: Comparative analysis of amino acids (AA) between patients with MG, MS, and within the control group allowed for the identification of statistically significant differences in the amino acid profile. Comparing the patients (patients with MS and MG) with the control group, and after taking the results of multiple tests into account, it was observed that amino acids such as ARG, PRO, TRP, CIT were significantly different between the groups. When considering the comparison between the AA concentrations in MS and MG patients, we found three AAs that were significantly different in the MS and MG groups, after correcting for multiple testing (CIT, GABA, and AAA). Higher concentrations of amino acids that showed significant differences were observed in patients with myasthenia gravis. Conclusions: Our results have indicated AAs that may prove valuable for improving the diagnostics of MS and MG patients. To better assess the potential utility of these markers, their performance requires further validation in a larger study group and limitation of possible confounding factors, e.g., medications and diet. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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12 pages, 1774 KiB

Open AccessCase Report

Comprehensive GC-MS Measurement of Amino Acids, Metabolites, and Malondialdehyde in Metformin-Associated Lactic Acidosis at Admission and during Renal Replacement Treatment

by Rene A. Posma, Stephan J. L. Bakker, Maarten W. Nijsten, Daan J. Touw and Dimitrios Tsikas

J. Clin. Med. 2024, 13(13), 3692; https://doi.org/10.3390/jcm13133692 - 25 Jun 2024

Cited by 2 | Viewed by 1719

Abstract

Metformin is the most widely used drug in type 2 diabetes. Regular metformin use has been associated with changes in concentrations of amino acids. In the present study, we used valid stable-isotope labeled GC-MS methods to measure amino acids and metabolites, including creatinine [...] Read more.

Metformin is the most widely used drug in type 2 diabetes. Regular metformin use has been associated with changes in concentrations of amino acids. In the present study, we used valid stable-isotope labeled GC-MS methods to measure amino acids and metabolites, including creatinine as well as malondialdehyde (MDA), as an oxidative stress biomarker in plasma, urine, and dialysate samples in a patient at admission to the intensive care unit and during renal replacement treatment because of metformin-associated lactic acidosis (MALA, 21 mM lactate, 175 µM metformin). GC-MS revealed lower concentrations of amino acids in plasma, normal concentrations of the nitric oxide (NO) metabolites nitrite and nitrate, and normal concentrations of MDA. Renal tubular reabsorption rates were altered on admission. The patient received renal replacement therapy over 50 to 70 h of normalized plasma amino acid concentrations and their tubular reabsorption, as well as the tubular reabsorption of nitrite and nitrate. This study indicates that GC-MS is a versatile analytical tool to measure different classes of physiological inorganic and organic substances in complex biological samples in clinical settings such as MALA. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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16 pages, 2306 KiB

Open AccessArticle

LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders

by Gregori Casals, María Antonieta Ballesteros, Angielys Zamora, Irene Martínez, Guillermo Fernández-Varo, Mireia Mora, Felicia A. Hanzu and Manuel Morales-Ruiz

Biomolecules 2024, 14(5), 558; https://doi.org/10.3390/biom14050558 - 6 May 2024

Cited by 5 | Viewed by 2543

Abstract

Introduction: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography–high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical [...] Read more.

Introduction: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography–high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. Methods: The present study aimed to validate a simple routine LC–HRMS method to quantify cortisol, cortisone, 6β-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. Results: The LC-HRMS method involved liquid–liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6β-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). Conclusion: A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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17 pages, 2119 KiB

Open AccessArticle

Quantitative Analysis of Cenobamate and Concomitant Anti-Seizure Medications in Human Plasma via Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry

by Linda Molteni, Bruno Charlier, Albino Coglianese, Viviana Izzo, Giovanni Assenza, Pierantonio Menna, Ugo de Grazia and Annachiara D’Urso

Molecules 2024, 29(4), 884; https://doi.org/10.3390/molecules29040884 - 17 Feb 2024

Cited by 3 | Viewed by 2267

Abstract

Cenobamate (CNB) is a new anti-seizure medication (ASM) recently introduced in clinical practice after approval by the FDA and EMA for the add-on treatment of focal onset seizures in adult patients. Although its mechanism of action has not been fully understood, CNB showed [...] Read more.

Cenobamate (CNB) is a new anti-seizure medication (ASM) recently introduced in clinical practice after approval by the FDA and EMA for the add-on treatment of focal onset seizures in adult patients. Although its mechanism of action has not been fully understood, CNB showed promising clinical efficacy in patients treated with concomitant ASMs. The accessibility of CNB could pave a way for the treatment of refractory or drug-resistant epilepsies, which still affect at least one-third of the patients under pharmacological treatment. In this context, therapeutic drug monitoring (TDM) offers a massive opportunity for better management of epileptic patients, especially those undergoing combined therapy. Here, we describe the first fully validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for the quantification of CNB and concomitant ASMs in human plasma, with samples extracted either manually or by means of a liquid handler. Our method was validated according to the most recent ICH International Guideline M10 for Bioanalytical Method Validation and Study Sample Analysis. The method proved to be selective for CNB and displayed a linear range from 0.8 to 80 mg/L; no matrix effect was found (98.2 ± 4.1%), while intra-day and inter-day accuracy and precision were within the acceptance range. Also, CNB short- and long-term stability in plasma under different conditions was assessed. Leftover human plasma samples were employed as study samples for method validation. Our method proved to be highly sensitive and selective to quantify CNB and concomitant ASMs in human plasma; therefore, this method can be employed for a routinely TDM-based approach to support physicians in the management of an epileptic patient. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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12 pages, 2273 KiB

Open AccessArticle

HPLC-MS/MS Analysis for Sphingosine 1-Phosphate after the Dephosphorylation by Hydrogen Fluoride

by Hee-Jung Kim, Seo-Hyeon Jung, Shokhid Gulyamov, Hyun-Gy Lee, Oybek Boyjigitov and Yong-Moon Lee

Separations 2024, 11(1), 34; https://doi.org/10.3390/separations11010034 - 18 Jan 2024

Cited by 1 | Viewed by 2513

Abstract

Sphingosine 1-phosphate (S1P) is a signaling lipid molecule involved in various cellular processes. It is important to develop a quantitative method for S1P to determine endogenous levels and to investigate its functions. As S1P is a tiny lipid component of most biological samples, [...] Read more.

Sphingosine 1-phosphate (S1P) is a signaling lipid molecule involved in various cellular processes. It is important to develop a quantitative method for S1P to determine endogenous levels and to investigate its functions. As S1P is a tiny lipid component of most biological samples, highly sensitive analysis by LC-MS/MS is required. The main challenge in S1P analysis by chromatography is peak-broadening due to the presence of a polar phosphate and the fact that S1P is indeed a zwitterion itself. In this study, we used hydrogen fluoride (HF) to efficiently remove a phosphate and then analyzed the surrogate, sphingosine, as a sharp peak by LC-ESI-MS/MS. We optimized the dephosphorylation reaction in terms of temperature and reaction time. Multiple reaction monitoring (MRM) for a dephosphorylated form of S1P and C17-S1P as an internal standard at m/z transition 300.4 > 282.4 (quantification ion), 300.4 > 262.4 (qualification ion), 286.3 > 268.2 (internal standard) was conducted. This method was validated by essential parameters such as specificity, linearity, range, LOQ, LOD, accuracy, precision, and repeatability. To confirm this new method, we quantified S1P levels in various serum products (100.0~284.4 nM). In the sample pretreatment conditions for extracting S1P, the concern about potential sphingosine contamination in serum was negligible. The dephosphorylation efficiency by this method was about two-fold higher than that of alkaline phosphatase (APase). To apply the method in vivo, we analyzed S1P in plasma and kidney tissues obtained from a chronic kidney disease (CKD) mouse model. S1P levels were increased only in CKD kidney tissue but not in plasma. In conclusion, by applying the dephosphorylation step with HF, we established a new, sensitive LC-MS/MS quantitative method for S1P that can be applied to biological samples. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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22 pages, 10064 KiB

Open AccessCommunication

The Mechanism of Hepatic Encephalopathy Induced by Thioacetamide Based on Metabolomics and Proteomics: A Preliminary Study

by Honghui Guo, Guang Wang, Wei Huang, Lingrui Li, Yang Bai, Haifeng Wang and Lina Gao

Int. J. Mol. Sci. 2024, 25(1), 284; https://doi.org/10.3390/ijms25010284 - 24 Dec 2023

Cited by 3 | Viewed by 2412

Abstract

Hepatic encephalopathy (HE) is a central nervous system dysfunction syndrome caused by acute and chronic liver failure or various portal systemic shunt disorders. HE arises from metabolic disorder and excludes other known types of encephalopathy. HE is a major cause of death in [...] Read more.

Hepatic encephalopathy (HE) is a central nervous system dysfunction syndrome caused by acute and chronic liver failure or various portal systemic shunt disorders. HE arises from metabolic disorder and excludes other known types of encephalopathy. HE is a major cause of death in people with liver disease. Early diagnosis and timely treatment are key to improving HE prognosis. Herein, we established a model of HE and performed metabolomics to identify 50 significantly differential metabolites between the HE group and control group. The main metabolic pathways associated with these differential metabolites were the purine metabolism, pyrimidine metabolism, aminoacyl tRNA biosynthesis, and glucose metabolism. Through proteomics analysis, we identified 226 significantly differential proteins (52 up-regulated and 174 down-regulated). The main (Kyoto Encyclopedia of Genes and Genomes) enrichment pathways were the Staphylococcus aureus infection, vitamin digestion and absorption, and complement and coagulation cascades. Through the conjoint analysis of proteomics and metabolomics, the differentially present proteins and metabolites were found to be involved in vitamin digestion and absorption, and ferroptosis pathways. In HE, malondialdehyde was significantly elevated, but glutathione was significantly diminished, and the redox balance was destroyed, thus leading to changes in proteins’ levels associated with the ferroptosis pathway. In conclusion, this study preliminarily explored the molecular and metabolic mechanisms underlying HE. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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21 pages, 5889 KiB

Open AccessEditor’s ChoiceArticle

Variations in O-Glycosylation Patterns Influence Viral Pathogenicity, Infectivity, and Transmissibility in SARS-CoV-2 Variants

by Sherifdeen Onigbinde, Cristian D. Gutierrez Reyes, Mojibola Fowowe, Oluwatosin Daramola, Mojgan Atashi, Andrew I. Bennett and Yehia Mechref

Biomolecules 2023, 13(10), 1467; https://doi.org/10.3390/biom13101467 - 29 Sep 2023

Cited by 9 | Viewed by 3286

Abstract

The highly glycosylated S protein plays a vital role in host cell invasion, making it the principal target for vaccine development. Differences in mutations observed on the spike (S) protein of SARS-CoV-2 variants may result in distinct glycosylation patterns, thus influencing immunological evasion, [...] Read more.

The highly glycosylated S protein plays a vital role in host cell invasion, making it the principal target for vaccine development. Differences in mutations observed on the spike (S) protein of SARS-CoV-2 variants may result in distinct glycosylation patterns, thus influencing immunological evasion, infectivity, and transmissibility. The glycans can mask key epitopes on the S1 protein and alter its structural conformation, allowing the virus to escape the immune system. Therefore, we comprehensively characterize O-glycosylation in eleven variants of SARS-CoV-2 S1 subunits to understand the differences observed in the biology of the variants. In-depth characterization was performed with a double digestion strategy and an efficient LC-MS/MS approach. We observed that O-glycosylation is highly conserved across all variants in the region between the NTD and RBD, whereas other domains and regions exhibit variation in O-glycosylation. Notably, omicron has the highest number of O-glycosylation sites on the S1 subunit. Also, omicron has the highest level of sialylation in the RBD and RBM functional motifs. Our findings may shed light on how differences in O-glycosylation impact viral pathogenicity in variants of SARS-CoV-2 and facilitate the development of a robust vaccine with high protective efficacy against the variants of concern. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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26 pages, 5078 KiB

Open AccessArticle

Cisplatin Dependent Secretion of Immunomodulatory High Mobility Group Box 1 (HMGB1) Protein from Lung Cancer Cells

by Kevin P. Gillespie, Ross Pirnie, Clementina Mesaros and Ian A. Blair

Biomolecules 2023, 13(9), 1335; https://doi.org/10.3390/biom13091335 - 31 Aug 2023

Cited by 8 | Viewed by 2789

Abstract

High mobility group box 1 (HMGB1) is secreted from activated immune cells, necrotic cells, and certain cancers. Previous studies have reported that different patterns of post-translational modification, particularly acetylation and oxidation, mediate HMGB1 release and confer distinct extracellular HMGB1 signaling activity. Here we [...] Read more.

High mobility group box 1 (HMGB1) is secreted from activated immune cells, necrotic cells, and certain cancers. Previous studies have reported that different patterns of post-translational modification, particularly acetylation and oxidation, mediate HMGB1 release and confer distinct extracellular HMGB1 signaling activity. Here we report that cisplatin but not carboplatin induces secretion of HMGB1 from human A549 non-small cell lung cancer (NSCLC) cells. Cisplatin-mediated HMGB1 secretion was dose-dependent and was regulated by nuclear exportin 1 (XPO1) also known as chromosomal maintenance 1 (CRM1) rather than adenosine diphosphate (ADP)-ribosylation, acetylation, or oxidation. HMGB1, as well as lysine acetylation and cysteine disulfide oxidation of secreted HMGB1, were monitored by sensitive and specific assays using immunoprecipitation, stable isotope dilution, differential alkylation, and nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (nano-LC-PRM/HRMS). A major fraction of the HMGB1 secreted by low-dose cisplatin treatment of A549 NSCLC cells was found to be in the fully reduced form. In contrast, mainly oxidized forms of HMGB1 were secreted by dimethyl sulfoxide (DMSO)-mediated apoptosis. These findings suggest that inhibition of XPO1 could potentiate the anti-tumor activity of cisplatin by increasing the nuclear accumulation of HMGB1 protein, an inhibitor of cisplatin DNA-adduct repair. Furthermore, low-dose cisplatin therapy could modulate the immune response in NSCLC through the established chemokine activity of extracellular reduced HMGB1. This could potentially enhance the efficacy of subsequent immunotherapy treatment. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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13 pages, 1439 KiB

Open AccessArticle

Development and Validation of a New LC-MS/MS Bioanalytical Method for the Simultaneous Determination of Levodopa, Levodopa Methyl Ester, and Carbidopa in Human Plasma Samples

by Linda Molteni, Bruno Charlier, Viviana Izzo, Albino Coglianese, Valeria Conti, Roberto Eleopra, Roberto Cilia, Chiara Capelli, Annachiara D’Urso and Ugo de Grazia

Molecules 2023, 28(11), 4264; https://doi.org/10.3390/molecules28114264 - 23 May 2023

Cited by 5 | Viewed by 3868

Abstract

Levodopa (L-DOPA) treatment, combined with the administration of dopa-decarboxylase inhibitors (DDCIs), is still the most effective symptomatic treatment of Parkinson’s disease (PD). Although its efficacy in the early stage of the disease has been confirmed, its complex pharmacokinetics (PK) increases the variability of [...] Read more.

Levodopa (L-DOPA) treatment, combined with the administration of dopa-decarboxylase inhibitors (DDCIs), is still the most effective symptomatic treatment of Parkinson’s disease (PD). Although its efficacy in the early stage of the disease has been confirmed, its complex pharmacokinetics (PK) increases the variability of the intra-individual motor response, thus amplifying the risk of motor/non-motor fluctuations and dyskinesia. Moreover, it has been demonstrated that L-DOPA PK is strongly influenced by several clinical, therapeutic, and lifestyle variables (e.g., dietary proteins). L-DOPA therapeutic monitoring is therefore crucial to provide personalized therapy, hence improving drug efficacy and safety. To this aim, we have developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method to quantify L-DOPA, levodopa methyl ester (LDME), and the DDCI carbidopa in human plasma. The compounds were extracted by protein precipitation and samples were analyzed with a triple quadrupole mass spectrometer. The method showed good selectivity and specificity for all compounds. No carryover was observed, and dilution integrity was demonstrated. No matrix effect could be retrieved; intra-day and inter-day precision and accuracy values met the acceptance criteria. Reinjection reproducibility was assessed. The described method was successfully applied to a 45-year-old male patient to compare the pharmacokinetic behavior of an L-DOPA-based medical treatment involving commercially available Mucuna pruriens extracts and an LDME/carbidopa (100/25 mg) formulation. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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11 pages, 1309 KiB

Open AccessArticle

Development of a Highly Sensitive Hybrid LC/MS Assay for the Quantitative Measurement of CTLA-4 in Human T Cells

by Dong Wei, Kristin L. Horton, John Chen, Linlin Dong, Susan Chen, Kojo Abdul-Hadi, Ting Ting Zhang, Cierra N. Casson, Michael Shaw, Tsubasa Shiraishi, Brandon Wilkinson, Chengjie Ji and Mark G. Qian

Molecules 2023, 28(8), 3311; https://doi.org/10.3390/molecules28083311 - 8 Apr 2023

Cited by 3 | Viewed by 2791

Abstract

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a check point protein expressed on the surface of T cells and plays a central role in regulating the immune response. In recent years, CTLA-4 has become a popular target for cancer immunotherapy in which blocking CTLA-4 [...] Read more.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a check point protein expressed on the surface of T cells and plays a central role in regulating the immune response. In recent years, CTLA-4 has become a popular target for cancer immunotherapy in which blocking CTLA-4 can restore T-cell function and enhance the immune response against cancer. Currently, there are many CTLA-4 inhibitors in a variety of modalities, including cell therapies, which are being developed in both preclinical and clinical stages to further harness the potential of the target for the treatment of certain types of cancer. In drug discovery research, measuring the level of CTLA-4 in T cells is important for drug discovery and development because it provides key information for quantitative assessment of the pharmacodynamics, efficacy, and safety of the CTLA-4-based therapies. However, to our best knowledge, there is still no report of a sensitive, specific, accurate, and reliable assay for CTLA-4 measurement. In this work, an LC/MS-based method was developed to measure CTLA-4 in human T cells. The assay demonstrated high specificity with an LLOQ of 5 copies of CTLA-4 per cell when using 2.5 million T cells for analysis. As shown in the work, the assay was successfully used to measure CTLA-4 levels in subtype T-cell samples from individual healthy subjects. The assay could be applied in supporting the studies of CTLA-4-based cancer therapies. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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21 pages, 5552 KiB

Open AccessArticle

Stability Determination of Intact Humanin-G with Characterizations of Oxidation and Dimerization Patterns

by Mustafa Ozgul, Anthony B. Nesburn, Nader Nasralla, Benjamin Katz, Enes Taylan, Baruch D. Kuppermann and Maria Cristina Kenney

Biomolecules 2023, 13(3), 515; https://doi.org/10.3390/biom13030515 - 11 Mar 2023

Cited by 2 | Viewed by 2122

Abstract

Humanin is the first identified mitochondrial-derived peptide. Humanin-G (HNG) is a variant of Humanin that has significantly higher cytoprotective properties. Here, we describe the stability features of HNG in different conditions and characterize HNG degradation, oxidation, and dimerization patterns over short-term and long-term [...] Read more.

Humanin is the first identified mitochondrial-derived peptide. Humanin-G (HNG) is a variant of Humanin that has significantly higher cytoprotective properties. Here, we describe the stability features of HNG in different conditions and characterize HNG degradation, oxidation, and dimerization patterns over short-term and long-term periods. HNG solutions were prepared in high-performance liquid chromatography (HPLC) water or MO formulation and stored at either 4 °C or 37 °C. Stored HNG samples were analyzed using HPLC and high-resolution mass spectrometry (HRMS). Using HPLC, full-length HNG peptides in HPLC water decreased significantly with time and higher temperature, while HNG in MO formulation remained stable up to 95% at 4 °C on day 28. HNG peptides in HPLC water, phosphate-buffered saline (PBS) and MO formulation were incubated at 37 °C and analyzed at day 1, day 7 and day 14 using HRMS. Concentrations of full-length HNG peptide in HPLC water and PBS declined over time with a corresponding appearance of new peaks that increased over time. These new peaks were identified to be singly oxidized HNG, doubly oxidized HNG, homodimerized HNG, singly oxidized homodimerized HNG, and doubly oxidized homodimerized HNG. Our results may help researchers improve the experimental design to further understand the critical role of HNG in human diseases. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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15 pages, 1008 KiB

Open AccessArticle

Clinical Evaluation Based on a New Approach to Improve the Accuracy of 4β-Hydroxycholesterol Measurement as a Biomarker of CYP3A4 Activity

by Yuki Taya, Mari Mizunaga, Shunsuke Nakao, Mirinthorn Jutanom, Naoki Shimizu, Yukihiro Nomura and Kiyotaka Nakagawa

Molecules 2023, 28(4), 1576; https://doi.org/10.3390/molecules28041576 - 7 Feb 2023

Cited by 2 | Viewed by 2751

Abstract

This study examines 4β-Hydroxycholesterol (4β-HC), which is considered to be a potential marker for the CYP3A4 induction of new chemical entities (NCEs) in drug development. To ensure the use of 4β-HC as a practical biomarker, it is necessary to accurately measure 4β-HC and [...] Read more.

This study examines 4β-Hydroxycholesterol (4β-HC), which is considered to be a potential marker for the CYP3A4 induction of new chemical entities (NCEs) in drug development. To ensure the use of 4β-HC as a practical biomarker, it is necessary to accurately measure 4β-HC and demonstrate that CYP3A4 induction can be appropriately assessed, even for weak inducers. In clinical trials of NCEs, plasma is often collected with various anticoagulants, in some cases, the plasma is acidified, then stored for an extended period. In this study, we examined the effects of these manipulations on the measurement of 4β-HC, and based on the results, we optimized the plasma collection and storage protocols. We also found that a cholesterol oxidation product is formed when plasma is stored, and by monitoring the compound, we were able to identify when plasma was stored inappropriately. After evaluating the above, clinical drug–drug interaction (DDI) studies were conducted using two NCEs (novel retinoid-related orphan receptor γ antagonists). The weak CYP3A4 induction by the NCEs (which were determined based on a slight decline in the systemic exposure of a probe substrate (midazolam)), was detected by the significant increase in 4β-HC levels (more specifically, 4β-HC/total cholesterol ratios). Our new approach, based on monitoring a cholesterol oxidation product to identify plasma that is stored inappropriately, allowed for the accurate measurement of 4β-HC, and thus, it enabled the evaluation of weak CYP3A4 inducers in clinical studies without using a probe substrate. Full article

(This article belongs to the Topic Chromatography–Mass Spectrometry Analysis in Biomedical Research and Clinical Laboratory)

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