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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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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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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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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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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.
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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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Comprehensive ESI-Q TRAP-MS/MS based characterization of metabolome of two mango (Mangifera indica L) cultivars from China.
Tan, L, Jin, Z, Ge, Y, Nadeem, H, Cheng, Z, Azeem, F, Zhan, R
Scientific reports. 2020;(1):20017
Abstract
Polyphenols based bioactive compounds from vegetables and fruits are known for impressive antioxidant activity. Ingestion of these antioxidants may promote human health against cardiovascular diseases and cancer. Mango is a popular tropical fruit with special taste, high nutritional value and health-enhancing metabolites. The aim was to investigate the diversity of phytochemicals between two mango cultivars of china at three stages of fruit maturity. We used ESI-QTRAP-MS/MS approach to characterize comprehensively the metabolome of two mango cultivars named Hongguifei (HGF) and Tainong (TN). HPLC was used to quantify selected catechin based phenolic compounds. Moreover, real-time qPCR was used to study the expression profiles of two key genes (ANR and LAR) involved in proanthocyanidin biosynthesis from catechins and derivatives. A total of 651 metabolites were identified, which include at least 257 phenolic compounds. Higher number of metabolites were differentially modulated in peel as compared to pulp. Overall, the relative quantities of amino acids, carbohydrates, organic acids, and other metabolites were increased in the pulp of TN cultivar. While the contents of phenolic compounds were relatively higher in HGF cultivar. Moreover, HPLC based quantification of catechin and derivatives exhibited cultivar specific variations. The ANR and LAR genes exhibited an opposite expression profile in both cultivars. Current study is the first report of numerous metabolites including catechin-based derivatives in mango fruit. These findings open novel possibilities for the use of mango as a source of bioactive compounds.
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Retention Time Prediction and Protein Identification.
Henneman, A, Palmblad, M
Methods in molecular biology (Clifton, N.J.). 2020;:115-132
Abstract
In bottom-up proteomics, proteins are typically identified by enzymatic digestion into peptides, tandem mass spectrometry and comparison of the tandem mass spectra with those predicted from a sequence database for peptides within measurement uncertainty from the experimentally obtained mass. Although now decreasingly common, isolated proteins or simple protein mixtures can also be identified by measuring only the masses of the peptides resulting from the enzymatic digest, without any further fragmentation. Separation methods such as liquid chromatography and electrophoresis are often used to fractionate complex protein or peptide mixtures prior to analysis by mass spectrometry. Although the primary reason for this is to avoid ion suppression and improve data quality, these separations are based on physical and chemical properties of the peptides or proteins and therefore also provide information about them. Depending on the separation method, this could be protein molecular weight (SDS-PAGE), isoelectric point (IEF), charge at a known pH (ion exchange chromatography), or hydrophobicity (reversed phase chromatography). These separations produce approximate measurements on properties that to some extent can be predicted from amino acid sequences. In the case of molecular weight of proteins without posttranslational modifications this is straightforward: simply add the molecular weights of the amino acid residues in the protein. For IEF, charge and hydrophobicity, the order of the amino acids, and folding state of the peptide or protein also matter, but it is nevertheless possible to predict the behavior of peptides and proteins in these separation methods to a degree which renders such predictions useful. This chapter reviews the topic of using data from separation methods for identification and validation in proteomics, with special emphasis on predicting retention times of tryptic peptides in reversed-phase chromatography under acidic conditions, as this is one of the most commonly used separation methods in bottom-up proteomics.
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Development and validation of samples stabilization strategy and LC-MS/MS method for simultaneous determination of clevidipine and its primary metabolite in human plasma: Application to clinical pharmacokinetic study in Chinese healthy volunteers.
Zhang, Y, Zhao, S, Zhou, H, Ding, L
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 2020;:122448
Abstract
A feasible LC-MS/MS method with reliable stabilizers consisted of sodium fluoride, ascorbic acid and formic acid was developed and validated for the determination of clevidipine and its primary metabolite (H152/81) in human plasma. Sodium fluoride existing in the vacutainer tubes was used to inhibit esterase activity to protect the clevidipine from hydrolysis as soon as blood was collected. Ascorbic acid and formic acid were added to the separated plasma samples to avoid the oxidation and further hydrolysis of clevidipine and H152/81. The further sample preparation was accomplished through a single step liquid-liquid extraction (LLE) by ethyl acetate. The chromatography separation was carried out on an ACE Excel 3 μm SuperC18 (2.1 × 50 mm, id, ACE, United Kingdom) column with gradient elution using 10 mM ammonium acetate water solution and methanol as the mobile phase. Detection was performed in the negative ion electrospray ionization mode using multiple reaction monitoring (clevidipine: m/z 454.1 → 234.0; clevidipine-d7: m/z 461.1 → 240.1; H152/81: m/z 354.0 → 208.0; H152/81-13CD3: m/z 358.0 → 212.0). The method exhibited good linearity over the concentration ranges of 0.100 to 40.0 ng/mL for clevidipine and 5.00 to 400 ng/mL for H152/81. The intra- and inter-batch precision and accuracy of clevidipine and H152/81 were all within the acceptable criteria. The method was successfully applied to a pharmacokinetic study of clevidipine and H152/81 in healthy Chinese volunteers following 8 mg/h intravenous infusion of clevidipine butyrate injectable emulsion for 0.5 h. The results showed that clevidipine was rapidly eliminated with a short half-life time of 0.244 ± 0.125 h and a maximum concentration of 25.2 ± 7.09 ng/mL. H152/81 was detectable in the plasma samples up to 48.5 h with a half-life time of 10.7 ± 2.30 h and a maximum plasma concentration of 301 ± 38.1 ng/mL.