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1.
Thermodynamics and Reaction Mechanisms for Decomposition of a Simple Protonated Tripeptide, H+GGA: From H+GGG to H+GAG to H+GGA.
Mookherjee, A, Armentrout, PB
Journal of the American Society for Mass Spectrometry. 2022;(2):355-368
Abstract
We present a thorough characterization of fragmentations observed in threshold collision-induced dissociation (TCID) experiments of protonated glycylglycylalanine (H+GGA) with Xe using a guided ion beam tandem mass spectrometer. Kinetic energy dependent cross sections for nine ionic products were obtained and analyzed to provide 0 K barriers for the five primary products, [b2]+, [y1 + 2H]+, [b3]+, [y2 + 2H]+, and [a1]+; and four secondary products, [a2]+, [a3]+, high-energy [y1 + 2H]+, and CH3CHNH2+, after accounting for multiple ion-molecule collisions, the internal energy of reactant ions, unimolecular decay rates, competition between channels, and sequential dissociations. Relaxed potential energy surface scans performed at the B3LYP-GD3BJ/6-311+G(d,p) level of theory are used to identify transition states (TSs) and intermediates of the five primary and three secondary products (with the mechanism of the other secondary product previously established). Geometry optimizations and single point energy calculations of reactants, products, intermediates, and TSs were performed at several levels of theory. These theoretical energies are compared with experimental threshold energies and found to give reasonable agreement, with B3LYP-GD3BJ and M06-2X levels of theory performing slightly better than MP2 and better than B3LYP. The results obtained here are compared with previous results for decomposition of H+GGG and H+GAG to probe the effect of changing the amino acid sequence. Methylation in H+GGA has a significant effect on the competition between the primary sequence products, [b2]+ and [y1 + 2H]+, suppressing the [b2]+ cross section by raising its threshold energy, while enhancing that of [y1 + 2H]+ by lowering its threshold energy.
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2.
Evaluation of Protein Purification Techniques and Effects of Storage Duration on LC-MS/MS Analysis of Archived FFPE Human CRC Tissues.
Rossouw, SC, Bendou, H, Blignaut, RJ, Bell, L, Rigby, J, Christoffels, A
Pathology oncology research : POR. 2021;:622855
Abstract
To elucidate cancer pathogenesis and its mechanisms at the molecular level, the collecting and characterization of large individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardized methods of formalin fixation and paraffin embedment, these archived FFPE tissues are important collections of pathology material that include patient metadata, such as medical history and treatments. FFPE blocks can be stored under ambient conditions for decades, while retaining cellular morphology, due to modifications induced by formalin. However, the effect of long-term storage, at resource-limited institutions in developing countries, on extractable protein quantity/quality has not yet been investigated. In addition, the optimal sample preparation techniques required for accurate and reproducible results from label-free LC-MS/MS analysis across block ages remains unclear. This study investigated protein extraction efficiency of 1, 5, and 10-year old human colorectal carcinoma resection tissue and assessed three different gel-free protein purification methods for label-free LC-MS/MS analysis. A sample size of n = 17 patients per experimental group (with experiment power = 0.7 and α = 0.05, resulting in 70% confidence level) was selected. Data were evaluated in terms of protein concentration extracted, peptide/protein identifications, method reproducibility and efficiency, sample proteome integrity (due to storage time), as well as protein/peptide distribution according to biological processes, cellular components, and physicochemical properties. Data are available via ProteomeXchange with identifier PXD017198. The results indicate that the amount of protein extracted is significantly dependent on block age (p < 0.0001), with older blocks yielding less protein than newer blocks. Detergent removal plates were the most efficient and overall reproducible protein purification method with regard to number of peptide and protein identifications, followed by the MagReSyn® SP3/HILIC method (with on-bead enzymatic digestion), and lastly the acetone precipitation and formic acid resolubilization method. Overall, the results indicate that long-term storage of FFPE tissues (as measured by methionine oxidation) does not considerably interfere with retrospective proteomic analysis (p > 0.1). Block age mainly affects initial protein extraction yields and does not extensively impact on subsequent label-free LC-MS/MS analysis results.
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3.
High-throughput LC-MS method to investigate postprandial lipemia: considerations for future precision nutrition research.
Mucinski, JM, Vena, JE, Ramos-Roman, MA, Lassman, ME, Szuszkiewicz-Garcia, M, McLaren, DG, Previs, SF, Shankar, SS, Parks, EJ
American journal of physiology. Endocrinology and metabolism. 2021;(4):E702-E715
Abstract
Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate postmeal handling of dietary lipids in humans are limited. This study tested a new method to track dietary lipid appearance using a stable isotope tracer (2H11-oleate) in liquid meals containing three levels of fat [low fat (LF), 15 g; moderate fat (MF), 30 g; high fat (HF), 60 g]. Meals were fed to 12 healthy men [means ± SD, age 31.3 ± 9.2 yr, body mass index (BMI) 24.5 ± 1.9 kg/m2] during four randomized study visits; the HF meal was administered twice for reproducibility. Blood was collected over 8 h postprandially, triglyceride (TG)-rich lipoproteins (TRL), and particles with a Svedberg flotation rate >400 (Sf > 400, n = 8) were isolated by ultracentrifugation, and labeling of two TG species (54:3 and 52:2) was quantified by LC-MS. Total plasma TRL-TG concentrations were threefold greater than Sf > 400-TG. Both Sf > 400- and TRL-TG 54:3 were present at higher concentrations than 52:2, and singly labeled TG concentrations were higher than doubly labeled. Furthermore, TG 54:3 and the singly labeled molecules demonstrated higher plasma absolute entry rates differing significantly across fat levels within a single TG species (P < 0.01). Calculation of fractional entry showed no significant differences in label handling supporting the utility of either TG species for appearance rate calculations. These data demonstrate the utility of labeling research meals with stable isotopes to investigate human postprandial lipemia while simultaneously highlighting the importance of examining individual responses. Meal type and timing, control of prestudy activities, and effects of sex on outcomes should match the research goals. The method, optimized here, will be beneficial to conduct basic science research in precision nutrition and clinical drug development.NEW & NOTEWORTHY A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.
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Simultaneous determination of 5-hydroxytryptophan and 3-O-methyldopa in dried blood spot by UPLC-MS/MS: A useful tool for the diagnosis of L-amino acid decarboxylase deficiency.
Di Carlo, E, Santagata, S, Sauro, L, Tolve, M, Manti, F, Leuzzi, V, Angeloni, A, Carducci, C
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 2021;:122999
Abstract
5-hydroxytryptophan (5HTP) and 3-O-methyldopa (3OMD) are CSF diagnostic biomarkers of the defect of aromatic L-amino acid decarboxylase (AADC), a rare inherited disorder of neurotransmitter synthesis which, if untreated, results in severely disabling neurological impairment. In the last few years, different methods to detect 3OMD in dried blood spot (DBS) were published. We developed and validated a fast and specific diagnostic tool to detect 5HTP alongside 3OMD. After extraction from DBS, 3OMD and 5HTP were separated by ultra-performance liquid chromatography (UPLC) and detected by tandem mass spectrometry (MS/MS). Instrument parameters were optimized to obtain the best sensitivity and specificity. Chromatographic separation was accomplished in 13 min. The limit of detection was 2.4 and 1.4 nmol/L of blood for 3OMD and 5HTP respectively, and response was linear over the blood range of 25-5000 nmol/L. Between-run imprecision was less than 9% for 3OMD and <13% for 5HTP. An age-specific continuous reference range was established, revealing a marked and continuous 3OMD decline with aging. The effect of age on 5HTP was less evident, showing only a slight decrease with age after the first week of life. A marked increase of both 3OMD and 5HTP was found in four patients affected by AADC deficiency (1780.6 ± 773.1 nmol/L, rv 71.0-144.9; and 94.8 ± 19.0 nmol/L, rv 15.2-42.8, respectively) while an isolated increase of 3OMD (6159.6 ± 3449.1 nmol/L, rv 73.2-192.2) was detected in three subjects affected by inherited disorders of dopamine synthesis under levodopa/carbidopa treatment (a marginal increase of 5HTP was detected in one of them). Simultaneous measurement of 5HTP and 3OMD in DBS leads to an improvement in specificity and sensitivity for the biochemical diagnosis of AADC deficiency.
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In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA.
Nyimanu, D, Kay, RG, Kuc, RE, Brown, AJH, Gribble, FM, Maguire, JJ, Davenport, AP
Peptides. 2021;:170642
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Abstract
BACKGROUND Elabela/Toddler (ELA) is a novel endogenous ligand of the apelin receptor, whose signalling has emerged as a therapeutic target, for example, in cardiovascular disease and cancer. Shorter forms of ELA-32 have been predicted, including ELA-21 and ELA-11, but metabolism and stability of ELA-32 in humans is poorly understood. We, therefore, developed an LC-MS/MS assay to identify ELA-32 metabolites in human plasma and tissues. METHOD Human kidney homogenates or plasma were incubated at 37 °C with ELA-32 and aliquots withdrawn over 2-4 h into guanidine hydrochloride. Proteins were precipitated and supernatant solid-phase extracted. Peptides were extracted from coronary artery, brain and kidney by immunoprecipitation or solid-phase extraction following acidification. All samples were reduced and alkylated before analysis on an Orbitrap mass spectrometer in high and nano flow mode. RESULTS The half-life of ELA-32 in plasma and kidney were 47.2 ± 5.7 min and 44.2 ± 3 s, respectively. Using PEAKS Studio and manual data analysis, the most important fragments of ELA-32 with potential biological activity identified were ELA-11, ELA-16, ELA-19 and ELA-20. The corresponding fragments resulting from the loss of C-terminal amino acids were also identified. Endogenous levels of these peptides could not be measured, as ELA peptides are prone to oxidation and poor chromatographic peaks. CONCLUSIONS The relatively long ELA plasma half-life observed and identification of a potentially more stable fragment, ELA-16, may suggest that ELA could be a better tool compound and novel template for the development of new drugs acting at the apelin receptor.
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Interpretation of Tandem Mass Spectra of Posttranslationally Modified Peptides.
Bunkenborg, J, Matthiesen, R
Methods in molecular biology (Clifton, N.J.). 2020;:199-230
Abstract
Tandem mass spectrometry provides a sensitive means of analyzing the amino acid sequence of peptides and modified peptides by providing accurate mass measurements of precursor and fragment ions. Modern mass spectrometry instrumentation is capable of rapidly generating many thousands of tandem mass spectra and protein database search engines have been developed to match the experimental data to peptide candidates. In most studies there is a schism between discarding perfectly valid data and including nonsensical peptide identifications-this is currently managed by establishing a false discovery rate (FDR) but for modified peptides it calls for an understanding of tandem mass spectrometry data. Manual evaluation of the data and perhaps experimental cross-checking of the MS data can save many months of experimental work trying to do biological follow-ups based on erroneous identifications. Especially for posttranslationally modified peptides there is a need for careful consideration of the data because search algorithms seldom have been optimized for the identification of modified peptides and because there are many pitfalls for the unwary. This chapter describes some of the issues that should be considered when interpreting and validating tandem mass spectra and gives some useful tables to aid in this process.
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Rapid and sensitive determination of neomycin and kanamycin in measles, mumps, and rubella vaccine via high-performance liquid chromatography-tandem mass spectrometry using modified super-paramagnetic Fe3O4 nanospheres.
Hamidi, H, Zarrineh, M, Es-Haghi, A, Ghasempour, A
Journal of chromatography. A. 2020;:461343
Abstract
A simple magnetic dispersive solid-phase extraction (MDSPE) methodology based on mesoporous Fe3O4@ succinic acid nanospheres and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed to determine kanamycin (KNM) and neomycin (NEO) contents in Measles, Mumps, and Rubella (MMR) vaccine products. The monodispersed mesoporous Fe3O4 nanospheres with self-assembled carboxyl terminated shell have been prepared via a simple solvothermal method. These as-synthesized mesoporous Fe3O4 nanospheres showed a high magnetic saturation value (Ms = 46 emu g-1) and large specific surface area (111.12 m2 g-1) which made them potential candidates as sorbents in magnetic solid-phase extraction. The adsorption experimental data fitted well with the Freundlich-Langmuir isotherm and followed a pseudo-second-order kinetic model. Moreover influential parameters on extraction efficiency were investigated and optimized. Under optimal conditions, the limits of detection for KNM and NEO were 1.0 and 0.1 ng mL-1, respectively. Recovery assessments using real samples exhibited recoveries in the range of 96.0 ± 4.3 to 101.5 ± 7.1 %, with relative standard deviations of <10.7% (for intra- day) and <14.6% (for inter- day). The proposed method was successfully applied for different spiked and un-spiked MMR vaccine samples. The presented extraction method provides a fast, selective, robust and practical platform for the detection of KNM and NEO in MMR vaccine samples.
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A LC-MS/MS method for therapeutic drug monitoring of sorafenib, regorafenib and their active metabolites in patients with hepatocellular carcinoma.
Iacuzzi, V, Zanchetta, M, Gagno, S, Poetto, AS, Orleni, M, Marangon, E, Guardascione, M, Foltran, L, Posocco, B, Toffoli, G
Journal of pharmaceutical and biomedical analysis. 2020;:113358
Abstract
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sorafenib (SORA), its N-oxide active metabolite and of regorafenib (REGO) and its two active metabolites regorafenib N-oxide and N-desmethyl regorafenib N-oxide in hepatocellular carcinoma patients' plasma. A proper analytes' separation was obtained with Synergi Fusion RP column (4 μm, 80 Å, 50 × 2.0 mm) using a gradient elution of 10 mM ammonium acetate with 0.1% formic acid (mobile phase A) and methanol:isopropanol (90:10, v/v, mobile phase B) containing 0.1% formic acid. The analysis was then performed by electrospray ionization in negative mode coupled with a triple quadrupole mass spectrometry, API 4000QT, monitoring two transitions for each analyte, one for the quantification and the other for confirmation. The method could be easily applied to the clinical practice thanks to the short run (7 min), the low amount of patient plasma necessary for the analysis (5 μL) and the fast sample processing based on protein precipitation. The method was therefore fully validated according to FDA and EMA guidelines. The linearity was assessed (R2≥0.998) over the concentration ranges of 50-8000 ng/mL for SORA and REGO, and 30-4000 ng/mL for their metabolites, that appropriately cover the therapeutic plasma concentrations. The presented method also showed adequate results in terms of intra- and inter-day accuracy and precision (CV ≤ 7.2% and accuracy between 89.4% and 108.8%), recovery (≥85.5%), sensitivity, analytes stability under various conditions and the absence of the matrix effect. Once the validation was successfully completed, the method was applied to perform the Cmin quantification of SORA, REGO and their metabolites in 54 plasma samples collected from patients enrolled in a clinical study ongoing at the National Cancer Institute of Aviano.
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Cellulose cone tip as a sorbent material for multiphase electrical field-assisted extraction of cocaine from saliva and determination by LC-MS/MS.
Sousa, DVM, Pereira, FV, Nascentes, CC, Moreira, JS, Boratto, VHM, Orlando, RM
Talanta. 2020;:120353
Abstract
A porous and hydrophilic sorbent material was used in an extraction system, assisted by electric fields, for the extraction of cocaine in saliva and subsequent determination by ultra-high-performance liquid chromatography associated with sequential triple quadrupole mass spectrometry (UHPLC-MS/MS). The cellulose-based material was characterized by scanning electron microscopy, infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction. The time and voltage variables applied in the extraction process were investigated through a Doehlert experimental design, and with the best conditions found (35min and 300 V) some validation parameters were evaluated. The established working range was 1-100 μg L-1 (R2 > 0.99), and the detection and quantification limits determined were 0.3 and 0.8 μg L-1, respectively. Recoveries from 80 to 115% and coefficient of variation ≤15 and 16% for intra-day and inter-day assays, respectively, were obtained for sample concentrations of LOQ, 5, 25, and 75 μg L-1, indicating satisfactory accuracy and precision for the proposed method. In addition, the method presented no matrix effect, and the extraction efficiency was between 56 and 70%. The results showed that the material used has adequate physicochemical characteristics and can be applied as a sorbent and electrolyte support in multiphase extractions using electric fields.
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Integrated phytochemical analysis based on UHPLC-LTQ-Orbitrap and network pharmacology approaches to explore the potential mechanism of Lycium ruthenicum Murr. for ameliorating Alzheimer's disease.
Luo, Z, Yu, G, Chen, X, Liu, Y, Zhou, Y, Wang, G, Shi, Y
Food & function. 2020;(2):1362-1372
Abstract
Based on compelling experimental and clinical evidence, the fruit of Lycium ruthenicum Murr. (LRM), a unique traditional Tibetan medicine, exerts beneficial effects on ameliorating learning and memory deficits of Alzheimer's disease (AD) and other neurodegenerative disorders. However, the potential active constituents and biological mechanism of LRM are still unknown. In this study, the major chemical constituents of LRM were first analyzed by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap). A total of 35 constituents were confirmed or tentatively identified. Furthermore, the network-based pharmacological strategy was applied to clarify the molecular mechanism of LRM on AD based on the identified components. Totally, 143 major targets were screened and supposed to be effective players in alleviating AD. Then, the LRM chemicals-major LRM putative targets-major pathways network was constructed, implying potential biological function of LRM on AD. More importantly, 12 core genes which can be modulated by LRM were identified, and they may play a pivotal role in alleviating some major symptoms of AD. This study provided a scientific basis for further investigation and application of LRM, which demonstrated that the network pharmacology approach could be a powerful way for the mechanistic studies of folk medicines.