Energy-resolved MS Research Articles

Quantitative comparison of bile acid glucuronides sub-metabolome between intrahepatic cholestasis and healthy pregnant women.

Because of the pathological indication and the physiological functions, bile acids (BAs) have occupied the research hotspot in recent decades. Although extensive efforts have been paid onto BAs sub-metabolome characterization, as the subfamily, BA glucuronides (gluA-BAs) profile is seldom concerned. Here, we made efforts to develop a LC-MS/MS program enabling quantitative gluA-BAs sub-metabolome characterization and to explore the differential species in serum between intrahepatic cholestasis of pregnancy (ICP) patients and healthy subjects. To gain as many authentic gluA-BAs as possible, liver microsomes from humans, rats, and mice were deployed to conjugate glucuronyl group to authentic BAs through in vitro incubation. Eighty gluA-BAs were captured and subsequently served as authentic compounds to correlate MS/MS spectral behaviors to structural features using squared energy-resolved MS program. Optimal collision energy (OCE) of [M-H]->[M-H-176.1]- was jointly administrated by [M-H]- mass and glucuronidation site, and identical exciting energies corresponding to 50% survival rate of 1st-generation fragment ion (EE50) were observed merely when the aglycone of a gluA-BA was consistent with the suspected structure. Through integrating high-resolution m/z, OCE, and EE50 information to identify gluA-BAs in a BAs pool, 97 ones were found and identified, and further, quantitative program was built for all annotated gluA-BAs by assigning OCEs to [M-H]->[M-H-176.1]- ion transitions. Quantitative gluA-BAs sub-metabolome of ICP was different from that of the healthy group. More GCDCA-3-G, GDCA-3-G, TCDCA-7-G, TDCA-3-G, and T-β-MCA-3-G were distributed in the ICP group. Above all, this study not only offered a promising analytical tool for in-depth gluA-BAs sub-metabolome characterization, but also clarified gluA-BAs allowing the differentiation of ICP and healthy subjects.

Isomeric differentiation of bile acids using three‐dimensional MS2 spectrum

AbstractBile acids (BAs) currently occupy the research hotspot because of their unreplaceable physiological functions as well as their unique abilities to reflect the physiopathological status. It is always challenging to characterize BAs‐submetabolome using LC–MS/MS, primarily attributing to the high‐level structural diversity. The key obstructing confirmative identification is isomeric identification because of the inherent “isomer‐blind” disadvantage of MS/MS. The isomerism styles of BAs are broadly divided into: type‐I, 5α‐H and 5β‐H epimers; type‐II, α‐OH and β‐OH epimers; type‐III, C‐OH positional isomers; and type‐IV, hybrid isomers bearing two or more isomerism fashions. Herein, we aim to comprehensively pursue isomer‐selective clues for BAs through constructing three‐dimensional MS2 (3D‐MS2) spectrum that was accomplished by fortifying energy‐resolved MS (ER‐MS) program as the new dimension. The breakdown graphs of primary MS2 spectral signals composed of 3D‐MS2 spectrum namely full collision energy ramp (FCER)‐MS2 spectrum for each BA species after appropriate normalization, and seven isomeric BAs covering all isomerism styles were utilized as representative cases. After recording MS2 spectra with progressive CE levels, differences were observed for their FCER‐MS2 spectra. Diagnostic fragment ions (DFIs) occurred within type‐I, type‐II, type‐III or type‐IV isomers, and noteworthily, 2.02 Da neutral loss discriminated BAs containing cis‐6,7‐diol and trans‐6,7‐diol functional groups. Notably, the features such as the maximal relative ion intensity (RIImax) and optimal CE (OCE) enabled isomeric differentiation for all types, even type‐II and type‐III isomers. Above all, it is feasible to acquire in‐depth isomer‐selective MS/MS clues for BAs using FCER‐MS2 spectrum, regardless of the isomerism manner.

Widely quasi-quantitative analysis of both metabolites and tryptic peptides in animal-originated medicinal materials: Bufonis Venenum as a case

It is challenging to achieve in-depth quality analysis for animal-originated medicinal materials (AMMs) owing to containing abundant metabolites and proteins. RPLC–MS/MS intrinsically bears the ability to simultaneously monitor metabolites and peptides. Hence, its potential towards merging quasi-quantitative metabolomics and tryptic proteomics characterization is therefore assessed in current study, and a well-known AMM namely Bufonis Venenum (BV, Chinese name: Chansu) was employed as a proof-of-concept. Qualitative information of metabolites was acquired by RPLC–Qtof-MS and translated to plausible structures through careful “spectrum-to-structure” analysis. Bottom-up proteomics was conducted to characterize the tryptic peptidome using nanoLC–QExactive HF orbitrap-MS. Quantitative MS/MS parameters of either metabolites (72 ones) or tryptic peptides (28 unique peptides) were optimized using online energy-resolved MS and applied to configure RPLC–selected-reaction monitoring (RPLC–SRM) program. Ultimately, SRM response of each analyte was converted to quasi-content by serially diluting a so-called universal metabolome standard (UMS) solution and building regressive calibration curve set to achieve widely quasi-quantitative metabolomics and proteomics. Although being sourced from identical species, significant differences occurred among the metabolite and protein profiles between BV and toad skins, and bufadienolides (i.e., 3-(N-adipoyl-argininyl)-gamabufotalin/isomer) along with several tryptic peptides (i.e., ISGLIYEETR sourced from Histone H4) served as the primary differential variables. Above all, RPLC–SRM is a promising analytical tool for in-depth quality evaluation of AMMs, and more importantly, the workflow described here is a fit-for-purpose pipeline to merge quasi-quantitative metabolomics and bottom-up proteomics.

Widely quasi-quantitative analysis enables temporal bile acids-targeted metabolomics in rat after oral administration of ursodeoxycholic acid

Bile acid (BA) pool homeostasis can be disturbed when cholestasis, and ursodeoxycholic acid (UDCA) treatment is able to rectify the perturbed pool. Efforts were made here to propose a strategy enabling BAs-focused widely quantitative metabolomics and subsequently to lucubrate BA pool fluctuation trajectory in rats after dosing UDCA. A so-called universal metabolome standard (UMS) sample containing numerous natural BAs was constructed by involving various available BAs-enriched matrices. In-depth chemical characterization was conducted for UMS to capture as many BAs as possible. Diverse survey experiments were performed on Qtrap-MS to search BAs, and IT-TOF-MS was deployed to provide high-resolution m/z values for both precursor and fragment ion species. After structural annotation for 120 BAs, in total, online energy-resolved MS was subsequently programmed to pursue superior parameters for certain BAs, particularly BAs bearing two conjugated moieties. UMS was serially diluted to generate calibration sample set, and the regressive calibration curves, 120 ones in total, were responsible for converting each analyte response to quasi-content (1/dilution fold). The validated widely quasi-quantitative program was then applied to profile BA pool shift trajectories against time in UDCA- and vehicle-treated rats. Mild variations were observed for the quantitative pattern of BA pool from vehicle group, whereas UDCA could significantly shape BAs sub-metabolome. Significant increments occurred for the contents of not only the downstream BAs, but some upstream molecules in BA metabolic network. The shift levels of BAs were primarily governed by their distances away from UDCA. Fourteen differential BAs were involved for absolute quantitation to generate the concentration vs. time patterns, and dramatic upgradation occurred for most BAs, consolidating the variations transmitted from large-scale quasi-quantitative metabolomics. Above all, current study revealed the quasi-quantitative details of BA pool fluctuation initiated by UDCA-treatment, and more importantly, provided a promising approach for temporal BAs-targeted metabolomics.

Quality structural annotation for the metabolites of chlorogenic acid in rat

Chlorogenic acid (CA) serves as a principal contributor for the health benefit spectrum of tea; however, its metabolism pattern hasn’t been completely clarified. We attempted here to profile CA metabolism through fortifying isomer identification ability onto LC–MS/MS. Online energy-resolved MS was applied to configure full collision energy ramp-MS2 spectrum. Quantum structural calculation was undetaken to pursue the relationships between chemical structures and optimal collision energy/the maximum relative ion intensity. Thirty-seven metabolites were captured in biological matrices. Plausible structures were configured by applying the empirical mass fragmentation rules to m/z values and the summarized relationships were utilized to drive the plausible structures to confidence-enhanced identities. Hydrolysis, glucuronidation, sulfation, acyl migration, oxidation, and hydrogenation occupied primary metabolism channels. Together, the current study disclosed in depth the metabolism profile of CA and moreover, suggested a versatile analytical route for quality metabolite identification.

Full Collision Energy Ramp-MS2 Spectrum in Structural Analysis Relying on MS/MS.

Albeit frequently being overlooked, MS2 spectrum variation against collision energy (CE) implies auxiliary structural clues for m/z values. Online energy-resolved MS (ER-MS) provides the opportunity to acquire the trajectory of ion intensity against CE for any fragment ion of interest, thus exactly offering the desired momentum to empower the conventional MS2 spectrum at a certain CE forward to a full-CE ramp MS2 spectrum (FCER-MS2). Efforts were made here to construct an FCER-MS2 spectrum and to evaluate its potential toward structural analysis. Flavonoids were employed as a proof of concept. MS2 spectra of 76 compounds were recorded by LC-Q-Exactive-MS, and online ER-MS was subsequently programmed using LC-Qtrap-MS to build a breakdown graph for each obvious fragment ion. After defining the greatest value amongst all regressive apices as 100%, the normalized breakdown graphs comprised an FCER-MS2 spectrum for each compound. The FCER-MS2 spectrum contained the MS2 spectrum at any CE as well as optimal CE (OCE) and maximal relative ion intensity (RIImax) of each fragment ion. Except the pronounced isomeric discrimination potential, either OCE or RIImax reflected certain structural properties, such as aglycone, glycosidic bond, and hydroxy, methoxy, and glycosyl substituents. These rules were subsequently applied for flavonoid-focused characterization of a famous herbal medicine, namely Scutellariae Radix, and high-level structural annotation was accomplished for 75 flavonoids. Above all, the FCER-MS2 spectrum includes m/z, OCEs, and RIImax features, thus facilitating confidence-advanced structural analysis.

Simultaneous determination of eight tryptic peptides in musk using high-performance liquid chromatography coupled with tandem mass spectrometry

In comparison of herbal medicines, less attention has been paid onto animal medicines, partially attributing to the protein-enriched property. Particularly, it is still challenging to conduct quality evaluation for the animal medicines because of the lack of a fit-for-purpose analytical tool. Herein, an attempt was made to propose a workflow allowing the quality assessment of animal medicines by LC–MS/MS, and musk that is one of the most precious traditional Chinese medicines was employed as a representative case for utility illustration. After the extraction of protein from musk with a well-defined protocol, tryptic digestion was conducted to hydrolyze proteins into peptides, and the peptide-enriched sample was subjected to nanoLC–Orbitrap MS measurement. The tandem mass spectral dataset was retrieved in Human Swiss-Prot FASTA database, and the sequences together with the sources of 733 tryptic peptides, in total, were annotated. Because of the abundant distributions, eight peptides were chosen as the analytes for quantitative measurements, and their quantitative MS parameters, such as ion transitions and collision energies, were rapid optimized in an authentic compound-free manner using online energy-resolved MS (ER-MS). On the other side, the annotated peptides were structurally consolidated via synthesizing reference peptides. When the synthetic peptides were applied for parameter optimization with the authentic compound-dependent manner, the values were almost identical with those from online ER-MS measurements. After being validated with diverse assays, the developed method was applied for the simultaneous determination of eight peptides in 28 batches of musk samples, including captive (C1–C18) and wild ones (W1–W10). Significant differences took place for the content patterns of concerned tryptic peptides between the captive and wild musk samples. Trace distributions occurred for DVDAAYMNK in most batches. Captive samples were rich of QSLEASLAETEGR, TLLDIDNTR, and EVATNSELVQSGK, whereas wild samples were able to accumulate YLGYLEQLLR. Together, the present study provided a meaningful approach for the quality evaluation of musk, as well as other peptide-enriched animal medicines, even if the absences of authentic peptides.

Integrated Strategy Drives Direct Infusion-Tandem Mass Spectrometry as an Eligible Tool for Shotgun Pseudo-Targeted Metabolomics of Medicinal Plants.

Direct infusion (DI) has an extraordinary high-throughput advantage. Pseudo-targeted metabolomics (PTM) has been demonstrated integrating the merits of both nontargeted and targeted metabolomics. Herein, we attempted to implant DI into the PTM concept to configure a new strategy allowing shotgun PTM. First, a versatile MS/MSALL program was applied to acquire MS1 and MS2 spectra. Second, online energy-resolved MS (online ER-MS) was conducted to obtain breakdown graph as well as optimal collision energy (OCE) for each ion transition paired by precursor ion and the dominant product ion. Third, selected reaction monitoring (SRM) was responsible to output a quantitative dataset with a constant length. Moreover, breakdown graph also served as orthogonal structural evidence when matching MS2 spectra between DI-MS/MS and an in-house library to strengthen structural annotation confidence. To evaluate and illustrate the utility of the new strategy toward shotgun PTM of medicinal plants, in-depth chemome comparison was conducted within three Cistanche species, all of which are edible medicinal plants and playing essential roles for turning the deserts into the oases. A total of 185 variables participated in the quantitative measurement program. Each diagnostic ion pair was featured with an OCE. Significant species differences occurred, and echinacoside, acteoside, isoacteoside, 2'-acetyl-acteoside, tubuloside B, mannitol, sucrose, betaine, malate, as well as choline were found to be confirmative chemical markers offering primary contributions toward the species discrimination. After cross-validation with LC-MS/MS, DI-MS/MS fortified with the new strategy is an eligible tool for shotgun PTM, beyond Cistanche plants.

Confirmative Structural Annotation for Metabolites of (R)-7,3'-Dihydroxy-4'-methoxy-8-methylflavane, A Natural Sweet Taste Modulator, by Liquid Chromatography-Three-Dimensional Mass Spectrometry.

Flavonoids occupy the largest family of natural products and possess a broad spectrum of health benefits. Their metabolites are sometimes the truly effective molecules in vivo. It is still challenging, however, to unambiguously identify flavonoid metabolites using conventional LC-MS/MS. Herein, we aimed to pursue auxiliary structural clues to m/z values in both MS1 and MS2 spectra through LC coupled to three-dimensional MS (LC-3D MS). MS1, as the first dimension, was in charge of suggesting theoretical molecular formulas, MS2, the as second dimension, was responsible for offering substructures, and exactly, online energy-resolved MS (ER-MS), as the third dimension, provided optimal collision energies (OCEs) that reflected the linkage manners among the substructures. Metabolic characterization of a natural sweet taste modulator, namely, (R)-7,3'-dihydroxy-4'-methoxy-8-methylflavane (DHMMF), was conducted as a proof-of-concept. Extensive efforts, such as full MS1 and MS2 scans on IT-TOF-MS and predictive selected-reaction monitoring mode on Qtrap-MS, were made for in-depth metabolite mining. Seventeen metabolites (M1-M17) were captured from DHMMF-treated biological samples, including 17 (M1-M17), 10 (M4-M9, M11, M13, M14, and M16), and 2 (M5 and M10) metabolites from urine, plasma, and feces, respectively. Their structures were configured by integrating MS1, MS2, and OCE information. Except M10, all metabolites were new compounds. LC-MS/MS-guided chromatographic purification yielded three glucuronyl-conjugated metabolites (M5, M8, and M11), and NMR spectroscopic assays consolidated the structures transmitted from LC-3D MS. Demethylation, glucuronidation, and sulfation occurred as the primary metabolic pathways of DHMMF. Above all, LC-3D MS bridged LC-MS/MS from putatively structural annotation toward confidence-enhanced identification, beyond the metabolite characterization of flavonoids.

Online energy-resolved MS boosts the potential of LC-MS towards metabolite characterization of salidroside and tyrosol.

Although currently serving as the workhorse for metabolite characterization, one of the most challenging tasks for LC-MS is isomeric differentiation because isomers frequently yield identical quasi-molecular ions and fragmented ion species. Our previous studies have demonstrated that online energy-resolved MS (ER-MS) is an orthogonal technique for MS/MS experiments to facilitate isomeric identification. Herein, attempts were made for the in-depth characterization of the metabolic profiles of an effective natural product named salidroside (SA) in rats using LC coupled with three-dimensional mass spectrometry (LC-3D MS) that was configured by MS1, MS2 and online ER-MS as 1st, 2nd, and 3rd dimensions, respectively. Moreover, the metabolism characterization of its aglycone, namely, tyrosol (Try) was conducted in parallel to aid in proposing metabolic pathways. High-resolution MS1 and MS2 spectra were acquired by IT-TOF-MS, and subsequent data processing provided theoretical formula and sub-structures for each metabolite. Subsequently, online ER-MS was conducted for precursor > product ion transitions-of-interest to offer linkage information among the sub-structures via building breakdown graphs. As a result, ten (M1-10) and nine (M1, M2, and M5-11) metabolites were detected in SA- and Tyr-administrated biological samples, respectively, and their structures were qualitatively identified. Crucial metabolism occurred for either component. SA initially underwent hydrolysis to produce Tyr, and subsequently hydroxylation, oxidation, glucuronidation, and sulfation were observed as the primary metabolic pathways. To summarize, the metabolic fate of SA was understood in depth, and Tyr, as the hydrolytic product, was responsible for the occurrences of most metabolites (M1, M2, and M5-10). More importantly, identification confidences of the metabolites were significantly advanced by LC-3D MS, suggesting that it is eligible to serve as an integral part of the analyst's toolbox.

Retention Time and Optimal Collision Energy Advance Structural Annotation Relied on LC-MS/MS: An Application in Metabolite Identification of an Antidementia Agent Namely Echinacoside.

The structural annotation of metabolites now relies heavily on HR-MS/MS information, resulting in ambiguous identities in most cases. More auxiliary evidence is therefore desired to achieve confirmative identification. Herein, we made an attempt to involve retention time (tR) along with optimal collision energy (OCE) as the additionally structural clues, and the applicability validation was conducted via confidence-enhanced metabolite characterization of echinacoside, an antidementia drug candidate within clinical trials. Quantitative structure-retention relationships (QSRR) were modeled via assaying 184 authentic compounds on RPLC, HILIC, and serially coupled RPLC and HILIC (RPLC-HILIC). Online energy-resolved MS was developed to yield breakdown graphs for selected ion transitions, and OCE was demonstrated to be superior to CE50 toward pointedly denoting the bonds-of-interest. Nineteen metabolites (M1-M19) were confidently identified in biological samples from echinacoside-treated rats by analyzing m/z values first to yield empirical formulas and substructures, and tR and OCE subsequently contributed to sift the candidate structures. Structural identification was validated by oral administration of three relevant compounds in parallel and chromatographic purification as well. Above all, the integration of retention and dissociation behaviors enabled promoting one step forward for structural annotation confidences merely relied on HR-MS/MS.

Optimal collision energy is an eligible molecular descriptor to boost structural annotation: An application for chlorogenic acid derivatives-focused chemical profiling

Tandem mass spectrometry (MS/MS), in particular high-resolution MS/MS, is able to provide element compositions and substructures for the detected signals. However, it is still challenging to configure the whole structures via linking those substructures. Efforts were devoted here to propose and validate optimal collision energy (OCE) to be an auxiliary structural clue to mass-to-charge ratios (m/z), and online energy-resolved MS was developed to yield OCEs. Chlorogenic acid derivatives (CADs) were utilized as the proof-of-concept because diverse isomers usually initiated by the different linkage manners between the quinic acid/shikimic acid and cinnamoyl substituents(s), i.e. caffeoyl group, coumaroyl group, etc. Liquid chromatography–hybrid ion trap-time of flight MS (LC–IT-TOF–MS) was implemented to capture CADs in two well-known herbal medicines namely Lonicerae japonicae Flos and Inulae Flos. Afterwards, hybrid triple quadrupole-linear ion trap MS (Qtrap-MS) was deployed to acquire OCEs for the primary fragmentation pathways of all detected CADs through online energy-resolved MS. On the other side, structural calculations were conducted to figure out the relationships between OCEs and bond properties. Isomeric differences occurred for OCEs, and LC elution program as well as ionization parameters could not affect OCEs. Twenty-four and thirty-one CADs were hunted and putatively identified by LC–IT-TOF-MS in Lonicerae japonicae Flos and Inulae Flos, respectively, and the structural annotation was advanced by applying the OCE-bond property relationships. To verify the structures, CADs-of-interest were purified from Lonicerae japonicae Flos using an automated fraction collector and definitely identified with NMR spectroscopy. Exact consistence occurred for the structural identification of mono-caffeoylquinic acid isomers between LC–MS/MS and NMR analyses. Consequently, OCE is an inherent physicochemical parameter of a given compound and is an eligible structural descriptor to offset the ability of LC–MS/MS towards the chemical profiling of complex matrices.

Plasma analysis of inductively coupled impulse sputtering of Cu, Ti and Ni

Inductively coupled impulse sputtering (ICIS) is a new development in the field of highly ionised pulsed PVD processes. For ICIS the plasma is generated by an internal inductive coil, replacing the need for a magnetron. To understand the plasma properties, measurements of the current and voltage waveforms at the cathode were conducted. The ion energy distribution functions (IEDFs) were measured by energy resolved MS and plasma chemistry was analysed by OES and then compared to a model. The target was operated in pulsed DC mode and the coil was energised by pulsed RF power, with a duty cycle of 7.5%. At a constant pressure (14 Pa) the set peak RF power was varied from 1000–4000 W. The DC voltage to the target was kept constant at 1900 V. OES measurements have shown a monotonic increase in intensity with increasing power. Excitation and ionisation processes were single step for ICIS of Ti and Ni and multi-step for Cu. The latter exhibited an unexpectedly steep rise in ionisation efficiency with power. The IEDFs measured by MS show the material- and time-dependant plasma potential in the range of 10–30 eV, ideal for increased surface mobility without inducing lattice defects. A lower intensity peak, of high energetic ions, is visible at 170 eV during the pulse.

Energy-resolved technique for discovery and identification of malonyl-triterpene saponins inCaulophyllum robustumby UHPLC-electrospray Fourier transform mass spectrometry

Malonyl-triterpene saponins (MTSs) attract scientific attentions because of their structural diversities and valuable bioactivities. However, its thermal instability brings a huge amount of challenges for isolation and purification of this class of compounds. To our best knowledge, there has been no report on isolation and analysis of MTSs from genus Caulophyllum. In this study, a strategy combining data acquisition using an energy-resolved technique and the narrow widow extracted ion chromatograms as data mining method was developed for discovery and identification of MTSs in Caulophyllum robustum hair roots by ultra high liquid chromatography coupled to electrospray ionization Fourier transform mass spectrometry. The method was performed at an independent MS full scan using our bottom-up energies by in-source collision induced dissociations with 0, 25, 50 and 100 eV in both positive and negative modes. Precursor ion as well as fragment ion information was simultaneously collected from four energy-resolved MS spectra in a single run of 18 min. The fragmentation pathways of intact deprotonated, protonated and sodium ions of MTSs were proposed for the structural elucidation of Caulophyllum MTSs. A flowchart involving a stepwise procedure based on key fragments from ESI- /ESI+ -FT-MS(1, 1) to MS(1, 4) spectra was constructed for the identification of structural elements in the MTSs. As a result, a total of 23 MTSs were discovered and tentatively identified, which had not been reported from Caulophyllum species before. All of these were potentially new compounds. This study provides an excellent example for discovery and identification of MTSs in herb medicines. Copyright © 2016 John Wiley & Sons, Ltd.