In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and β2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits. The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20-40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied. Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method.

• Klíčová slova
• Anti-Doping analysis, Collision cross section, High resolution mass spectrometry, Ion mobility spectrometry, Ultra-high performance liquid chromatography,
• MeSH
• doping ve sportu * MeSH
• glukokortikoidy MeSH
• iontová mobilní spektrometrie * MeSH
• narkotika MeSH
• steroidy MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukokortikoidy MeSH
• narkotika MeSH
• steroidy MeSH

Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions. Graphical Abstract ᅟ.

• Klíčová slova
• Collisional cross sections, Density functional theory calculations, Ion mobility, Ion structures, Peptide ions, Polar effects,
• MeSH
• helium MeSH
• iontová mobilní spektrometrie * MeSH
• kationty MeSH
• konformace proteinů * MeSH
• peptidy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• helium MeSH
• kationty MeSH
• peptidy * MeSH

Electrophoretic mobility shift assay (EMSA) is a well-established technique to monitor interactions between biomolecules particularly DNA and proteins. Even though numerous variations of this method have been presented, challenges in the form of detection sensitivity and/or variations in the stability of the formed complex still remain. With advances in the area of nanomaterials improvements in EMSA have been also suggested. Recently, Zhang and Wang (Electrophoresis 2015, 36, 1011-1015) presented electrophoretic mobility shift method for determination of number of DNA molecules conjugated to quantum dots (QDs), which was further utilized for calculation of enzymatic activity, sequence specific DNA detection, and neutral molecule quantification.

• Klíčová slova
• DNA, Electrophoretic mobility shift assay, Protein, Quantum dots,
• MeSH
• DNA analýza   chemie   MeSH
• kvantové tečky analýza   chemie   MeSH
• nanokonjugáty analýza   chemie   MeSH
• retardační test metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• nanokonjugáty MeSH

Analysis of new psychoactive substances (NPS), which is essential for toxicological and forensic reasons, can be made complicated by the presence of isomers. Ion mobility has been used as a standalone technique or coupled to mass spectrometry to detect and identify NPS. However, isomer separation has so far chiefly relied on chromatography. Here we report on the determination of isomeric ratios using cyclic ion mobility-mass spectrometry without any chromatographic separation. Isomers were distinguished by mobility separation of lithium adducts. Alternatively, we used arrival time distribution (ATD) profiles that were characteristic of individual isomers and were acquired for protonated molecules or fragment ions. Both approaches provided comparable results. Calculations were used to determine the structures and collision cross sections of both protonated and lithiated isomers that accurately characterized their ion mobility properties. The applicability of ATD profiles to isomer differentiation was demonstrated using direct infusion and flow injection analysis with electrospray of solutions, as well as desorption electrospray of solid samples. Data processing was performed by applying multiple linear regression to the ATD profiles. Using the proposed ATD profile-based approach, the relationships between the determined and given content of isomers showed good linearity with coefficients of determination typically greater than 0.99. Flow injection analysis using an autosampler allowed us to rapidly determine isomeric ratios in a sample containing two isomeric pairs with a minor isomer of 10% (determined 9.3% of 3-MMC and 11.0% of 3-FMC in a mixture with buphedrone and 4-FMC). The proposed approach is not only useful for NPS, but also may be applicable to small isomeric molecules analyzed by ion mobility when complete separation of isomers is not achieved.

• MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• iontová mobilní spektrometrie * metody   MeSH
• isomerie MeSH
• lithium chemie   MeSH
• psychotropní léky * chemie   analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lithium MeSH
• psychotropní léky * MeSH

Silymarin, milk thistle (Silybum marianum) extract, contains a mixture of mostly isomeric bioactive flavonoids and flavonolignans that are extensively studied, especially for their possible liver-protective and anticancer effects. Because of the differing bioactivities of individual isomeric compounds, characterization of their proportion in a mixture is highly important for predicting its effect on health. However, because of silymarin's complexity, this is hardly feasible by common analytical techniques. In this work, ultraperformance liquid chromatography coupled with drift tube ion mobility spectrometry and quadrupole time-of-flight mass spectrometry was used. Eleven target silymarin compounds (taxifolin, isosilychristin, silychristins A and B, silydianin, silybins A and B, 2,3-cis-silybin B, isosilybins A and B and 2,3-dehydrosilybin) and five unknown flavonolignan isomers detected in the milk thistle extract were fully separated in a 14.5-min analysis run. All the compounds were characterized on the basis of their accurate mass, retention time, drift time, collision cross section and fragmentation spectra. The quantitative approach based on evaluation of the ion mobility data demonstrated lower detection limits, an extended linear range and total separation of interferences from the compounds of interest compared with the traditional approach based on evaluation of liquid chromatography-quadrupole time-of-flight mass spectrometry data. The following analysis of a batch of milk thistle-based food supplements revealed significant variability in the silymarin pattern, especially in the content of silychristin A and silybins A and B. This newly developed method might have high application potential, especially for the characterization of materials intended for bioactivity studies in which information on the exact silymarin composition plays a crucial role. Graphical Abstract.

• Klíčová slova
• Collision cross section, Drift tube ion mobility high-resolution mass spectrometry, Milk thistle, Silybum marianum, Silymarin, Ultraperformance liquid chromatography,
• MeSH
• flavonolignany analýza   izolace a purifikace   MeSH
• hmotnostní spektrometrie metody   MeSH
• iontová mobilní spektrometrie metody   MeSH
• isomerie MeSH
• ostropestřec mariánský chemie   MeSH
• silymarin analýza   izolace a purifikace   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• flavonolignany MeSH
• silymarin MeSH

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

• MeSH
• hmotnostní spektrometrie MeSH
• iontová mobilní spektrometrie * MeSH
• lidé MeSH
• peptidy MeSH
• proteiny MeSH
• proteomika * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• peptidy MeSH
• proteiny MeSH

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis. In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, TWCCSN2 values, and m/z ratios was constructed. Urine samples were analyzed using either a simple "dilute and shoot" procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and TWCCSN2 values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses.

• Klíčová slova
• Collision cross section, Doping analysis, High resolution mass spectrometry, Ion mobility spectrometry, Ultra-high performance liquid chromatography,
• MeSH
• anabolika * MeSH
• doping ve sportu * MeSH
• hmotnostní spektrometrie MeSH
• iontová mobilní spektrometrie MeSH
• odhalování abúzu drog MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anabolika * MeSH

Lipid A, a crucial component of lipopolysaccharides (LPS), plays a pivotal role in the pathogenesis of Gram-negative bacteria. Lipid A patterns are recognized by mammals and can induce immunostimulatory effects. However, the outcome of the interaction is highly dependent on the chemical composition of individual lipid A species. The diversity of potential fatty acyl and polar headgroup combinations in this complex saccharolipid presents a significant analytical challenge. Current mass spectrometry (MS)-based lipid A methods are focused on either direct matrix-assisted laser desorption/ionization (MALDI)-MS screening or comprehensive structural elucidation by tandem mass spectrometry (MS/MS) hyphenated with separation techniques. In this study, we developed an alternative workflow for rapid lipid A profiling covering the entire analysis pipeline from sample preparation to data analysis. This workflow is based on microextraction and subsequent MALDI-MS/MS analysis of uropathogenic Escherichia coli utilizing trapped ion mobility spectrometry (TIMS), followed by mzmine data processing. The additional TIMS dimension served for enhanced sensitivity, selectivity, and structural elucidation through mobility-resolved fragmentation via parallel accumulation-serial fragmentation (PASEF) in parallel reaction monitoring (prm)-mode. Furthermore, mzmine enabled automated MS/MS acquisition by adapting the spatial ion mobility-scheduled exhaustive fragmentation (SIMSEF) strategy for MALDI spot analysis. It also facilitated robust lipid A annotation through a newly developed extension of the rule-based lipid annotation module, allowing for the custom generation of lipid classes, including specific fragmentation rules. In this study, the first publication of lipid A species' collision cross section (CCS) values is reported, which will enhance high-confidence lipid A annotation in future studies.

• MeSH
• gramnegativní bakterie * chemie   MeSH
• iontová mobilní spektrometrie * metody   MeSH
• lipid A * analýza   chemie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• uropatogenní Escherichia coli * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lipid A * MeSH

OBJECTIVE: The aim of our study was to asses changes in the mobility of the whole urethra after successful TVT procedure. DESIGN: Prospective pilot study. SETTING: Obstet. Gynecol Department, General Teaching Hospital, 1st Medical Faculty, Charles University, Prague; EuroMISE Centre of the Charles University and Academy of Sciences, Prague, Czech Republic. METHODS: 101 women with proven stressed urinary incontinence were included in the study. After the TVT procedure 90 women were evaluated. As a part of the complex urogynecological investigation before surgery the transperineal ultrasound scan was performed in supine position, urinary bladder was filled to 300 ml. In the orthogonal system of coordinates the position and mobility of the whole urethra before surgery were assessed. Control examination was done 3-6 months after the surgery. The changes induced by the surgery were assessed. For the statistical evaluation t-test, Wilcox test, F test, Kruskal-Wallis test and ANOVA were used. RESULTS: Surgery significantly decreased the mobility of the whole parts of the urethra during maximal Valsalva, but the position at rest is not influenced. The women with high urethral mobility have high mobility after the surgery. The operation was more effect in patients with high mobility. Never the less the change of relative mobility is the same in all women. CONCLUSIONS: The information about the type of urethral mobility is important and may increase the success rate of TVT. Therefore the tension of the tape should be different for patients with different urethral mobility.

• MeSH
• lidé středního věku MeSH
• lidé MeSH
• pohyb MeSH
• stresová inkontinence moči chirurgie   MeSH
• uretra patofyziologie   MeSH
• urodynamika MeSH
• Valsalvův manévr MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH

There is a need to better understand lipid metabolism during mosquito ovarian development. Lipids are the major source of energy supporting ovarian follicles development in mosquitoes. In this paper, we describe the complementary use of stable isotope labeling (SIL) and high-resolution mass spectrometry-based tools for the investigation of de novo triglycerides (TG) and diglycerides (DG) during the ovarian previtellogenic (PVG) stage (4-6 days posteclosion) of female adult Aedes aegypti. Liquid chromatography coupled to high-resolution trapped ion mobility spectrometry-parallel accumulation sequential fragmentation-time-of-flight tandem mass spectrometry (LC-TIMS-PASEF-TOF MS/MS) allowed the separation and quantification of nonlabeled and 2H/13C-labeled TG and DG species. Three SIL strategies were evaluated (H2O/2H2O with 50:50 and 95:5 mixtures, 13C-sucrose, and 13C-glucose). Results showed wide applicability with no signs of lipid ovarian impairment by SIL induced toxicity. The analytical workflow based on LC-TIMS-TOF MS/MS provided high confidence and high reproducibility for lipid DG and TG identification and SIL incorporation based on their separation by retention time (RT), collision cross section (CCS), and accurate m/z. In addition, the SIL fatty acid chain incorporation was evaluated using PASEF MS/MS. The 2H/13C incorporation into the mosquito diet provided information on how TG lipids are consumed, stored, and recycled during the PVG stage of ovarian development.

• MeSH
• chromatografie kapalinová MeSH
• Culicidae * MeSH
• diglyceridy analýza   chemie   MeSH
• iontová mobilní spektrometrie MeSH
• izotopové značení MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• diglyceridy MeSH