We have developed an electron-activated dissociation (EAD) device with product isolation functionality for multistage tandem mass spectrometry (MSn). The EAD portion is a branched magneto-radio frequency ion trap with an electron beam source, and we attached a linear radio frequency quadrupole (RFQ) ion trap (or D trap) to one of the branches of the EAD device. Because the D trap was installed inside a strong ring permanent magnet, we made the linear quadrupole rods from para-magnetic stainless steel, which works as a magnetic shield. Using this D trap, we isolated a fragment produced by the first dissociation technique, which can be further fragmented by collision-induced dissociation (CID) or EAD. Using the new EAD-D trap, we demonstrated a near-complete structural identification of cardiolipins via an MS3 workflow with CID followed by electronically excited dissociation (EED) after isolation from the CID products. The number of double bonds, their positions, and cis/trans isomerism in each acyl group, as well as the regioisomerism of acyl chains, were fully identified; however, the only remaining structural ambiguity of the cardiolipins was the chirality at the central carbon of the central glycerol group. We identified 18 cardiolipins in Escherichia coli (E. coli) extract.

Lipidomic studies demand analytical platforms capable of capturing the structural diversity and complexity of lipid species with accuracy, precision, and speed. The Echo® MS+ system, an advanced acoustic ejection (AE) system coupled with a mass spectrometer (MS), offers a genuinely high-throughput, contactless workflow for comprehensive lipid profiling. In this study, we developed an automated high-throughput workflow based on a single-phase lipid extraction using 1-octanol, and methanol with 10mM ammonium formate as a carrier solvent, to measure lipid profiles covering 10 lipid classes, namely, phosphatidylcholine (PC), lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, triacylglycerol, diacylglycerol, sphingomyelin, cholesteryl ester and cholesterol from plasma samples. We optimized sample preparation protocols, solvent systems, and droplet ejection parameters to maintain sensitivity and minimize variability of lipid signals. The extraction recoveries for lipid class representatives, typically between 89% and 95%, were found to be comparable to those obtained with the traditional butanol/methanol extraction method. We also tested the extract integrity; 1-octanol extracts did not show any degradation of lipids after the storage for several days. We assessed the fast analysis AE-MS mode, operating at a speed of 4 seconds per sample, for the quantitation of targeted lipid panels. The signal reproducibility for the lipid class representatives was better than 6% and the linear response was confirmed across 2 orders of magnitude. The semi-quantitative profile from AE-MS analysis of a target panel of 14 ceramides in NIST SRM 1950 was consistent with the published data. Additionally, we investigated a 9-second-wide peak AE-MS mode, operating at a speed of 12 seconds per sample, for its utility in untargeted qualitative lipid analysis. We employed the data dependent high-resolution (HR) MS acquisition. Data analysis of plasma extracts involved a search for the 14 most common plasma lipid classes, and it resulted in a comprehensive lipid profile, consistently identifying 200 most abundant lipid molecular ions per polarity in all samples in 384-well plate; a total of 338 lipid were reported as sum compositions. The MS information was supplemented with lipid structure details and tentative molecular species assignments based on the corresponding MS/MS data. For example, 42 major PC lipids measured in HR MS were found to correspond to 69 PC molecular species. This new technique, combining the single-phase 1-octanol plasma extraction with AE-MS analysis offers a carryover-free workflow, enabling ultra-fast comprehensive lipid measurements using small nanolitre extracts and minimal solvent volumes.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

We report "plasma" electron detachment dissociation (EDD), a novel electron-activated dissociation (EAD) method for the fast sequencing of oligonucleotides with a high sequence coverage. To reduce the repulsive Coulombic force between the deprotonated oligonucleotides and the electron beam, we performed EDD in a neutral electron-nitrogen (N2+) plasma stored in a magneto radio-frequency ion trap. We confirmed that plasma EDD accomplished a high sequence coverage (100%) of RNA with 40 mers in the reaction time of 10 ms using the electron beam kinetic energy of 35 eV. This new technique was applied to various modifications in oligonucleotide therapeutics (ONTs). Phosphorothioate (PS) positions showed an extremely high dissociation efficiency, i.e., 100 times higher than the standard phosphate (PO) in DNA. Locked nucleotides did not show intensive dissociation in EDD; however, collision-induced dissociation (CID) helped sequence these portions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a ZenoTOF mass spectrometer equipped with the plasma EDD technique successfully identified impurities in degraded samples.

We report the progress on an electron-activated dissociation (EAD) device coupled to a quadrupole TOF mass spectrometer (QqTOF MS) developed in our group. This device features a new electron beam optics design allowing up to 100 times stronger electron currents in the reaction cell. The electron beam current reached the space-charge limit of 0.5 μA at near-zero electron kinetic energies. These advances enable fast and efficient dissociation of various analytes ranging from singly charged small molecules to multiply protonated proteins. Tunable electron energy provides access to different fragmentation regimes: ECD, hot ECD, and electron-impact excitation of ions from organics (EIEIO). The efficiency of the device was tested on a wide range of precursor charge states. The EAD device was installed in a QqTOF MS employing a novel trap-and-release strategy facilitating spatial mass focusing of ions at the center of the TOF accelerator. This technique increased the sensitivity 6-10 times and allows for the first time comprehensive structural lipidomics on an LC time scale. The system was evaluated for other compound classes such as intact proteins and glycopeptides. Application of hot ECD for the analysis of glycopeptides resulted in rich fragmentation with predominantly peptide backbone fragments; however, glycan fragments attributed to the ECD process were also observed. A standard small protein ubiquitin (8.6 kDa) was sequenced with 90% cleavage coverage at spectrum accumulation times of 100 ms and 98% at 800 ms. Comparable cleavage coverage for a medium-size protein (carbonic anhydrase: 29 kDa) could be achieved, albeit with longer accumulation times.

Compared to their linear counterparts, cyclic peptides show better biological activities, such as antibacterial, immunosuppressive, and anti-tumor activities, and pharmaceutical properties due to their conformational rigidity. However, cyclic peptides could form numerous putative metabolites from potential hydrolytic cleavages and their fragments are very difficult to interpret. These characteristics pose a great challenge when analyzing metabolites of cyclic peptides by mass spectrometry. This study was to assess and apply a software-aided analytical workflow for the detection and structural characterization of cyclic peptide metabolites. Insulin and atrial natriuretic peptide (ANP) as model cyclic peptides were incubated with trypsin/chymotrypsin and/or rat liver S9, followed by data acquisition using TripleTOF® 5600. Resultant full-scan MS and MS/MS datasets were automatically processed through a combination of targeted and untargeted peak finding strategies. MS/MS spectra of predicted metabolites were interrogated against putative metabolite sequences, in light of a, b, y and internal fragment series. The resulting fragment assignments led to the confirmation and ranking of the metabolite sequences and identification of metabolic modification. As a result, 29 metabolites with linear or cyclic structures were detected in the insulin incubation with the hydrolytic enzymes. Sequences of twenty insulin metabolites were further determined, which were consistent with the hydrolytic sites of these enzymes. In the same manner, multiple metabolites of insulin and ANP formed in rat liver S9 incubation were detected and structurally characterized, some of which have not been previously reported. The results demonstrated the utility of software-aided data processing tool in detection and identification of cyclic peptide metabolites.

A hybrid SWATH/MS and HR-SRM/MS acquisition approach using multiple unit mass windows and 100 u precursor selection windows has been developed to interface with a chromatographic lipid class separation. The method allows for the simultaneous monitoring of sum compositions in MS1 and up to 48 lipids in MS2 per lipid class. A total of 240 lipid sum compositions from five phospholipid classes could be monitored in MS2 (HR-SRM/MS) while there was no limitation in the number of analytes in MS1 (HR-SIM/MS). On average, 92 lipid sum compositions and 75 lipid species could be quantified in human plasma samples. The robustness and precision of the workflow has been assessed using technical triplicates of the subject samples. Lipid identification was improved using a combined qualitative and quantitative data processing based on prediction instead of library search. Lipid class specific extracted ion currents of precursors and the corresponding molecular species fragments were extracted based on the information obtained from lipid building blocks and a combinatorial strategy. The SWATH/MS approach with the post-acquisition processing is not limited to the analyzed phospholipid classes and can be applied to other analytes and samples of interest.

The specific positions of carbon–carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties of the lipid that ultimately inform its biological function(s). Contemporary liquid chromatography–mass spectrometry (MS) strategies based on electrospray ionization coupled to tandem MS can easily detect fatty acyl lipids but generally cannot reveal those specific site(s) of unsaturation. Herein, we describe a novel and versatile workflow whereby fatty acids are first converted to fixed charge N -(4-aminomethylphenyl)pyridinium (AMPP) derivatives and subsequently subjected to ozone-induced dissociation (OzID) on a modified triple quadrupole mass spectrometer. The AMPP modification enhances the detection of fatty acids introduced by direct infusion. Fragmentation of the derivatized fatty acids also provides diagnostic fragment ions upon collision-induced dissociation that can be targeted in precursor ion scans to subsequently trigger OzID analyses in an automated data-dependent workflow. It is these OzID analyses that provide unambiguous assignment of carbon–carbon double bond locations in the AMPP-derivatized fatty acids. The performance of this analysis pipeline is assessed in profiling the patterns of unsaturation in fatty acids within the complex biological secretion vernix caseosa . This analysis uncovers significant isomeric diversity within the fatty acid pool of this sample, including a number of hitherto unreported double bond positional isomers that hint at the activity of potentially new metabolic pathways.

In recent years, the biochemical study of lipids has transformed from slow multi-dimensional chromatographic separations and chemical derivatization strategies to higher throughput analysis by mass spectrometry. Advances in mass spectrometry have enabled in-depth lipidomic analyses with unparalleled qualitative and quantitative sensitivity. However, unambiguous identification and quantitation of lipid molecular species in total lipid extracts has proven to be difficult, primarily due to isobaric overlapping isobaric and isomeric species. There are greater than 100,000 lipid molecular species present in a typical biological lipid extract that occupy a narrow mass range (~400-1100 amu), making such overlap a significant problem.

The inability of current mass spectrometry techniques to differentiate phospholipid isomers results in a routine under-estimation of phospholipid molecular diversity in complex biological matrices. Recent technological advances in tandem mass spectrometry and ion activation are helping to overcome these limitations, but all rely on tandem mass spectrometry with unit mass-selection and suffer from co-isolation of isobaric or isomeric species. Accordingly, separation of phospholipid isomers and isobars prior to characterization is required to fully delve into the complexity of the lipidome. Here we present a novel two-stage workflow combining reversed-phase ultra-high performance liquid chromatography with ozone-induced dissociation (OzID) and combined-collision- and ozone-induced-dissociation ( COzID) that reduces spectral complexity and enables discrimination of lipid isomers and isobars. Application of this technique to the analysis of human red blood cell lipid extracts allowed the separation, or partial separation, of adduct ion and head group isobars as well as double bond and sn-positional isomers affording near complete structural characterization of low abundance lipids, e.g. PC 18:0/20:3(n-6), PS 18:0/20:4(n-6) and PS 20:4(n-6)/18:0 all observed at m/z 834.7. We also introduce a software plug-in that automatically annotates OzID mass spectra to assign the carbon-carbon double bond positions in lipids. This new workflow allows us to delve deeper into the lipidome and represents another valuable tool for the lipidomics toolbox. Crown Copyright (C) 2018 Published by Elsevier B.V. All rights reserved.

High-quality mass spectral libraries have become crucial in mass spectrometry-based metabolomics. Here, we investigate a workflow to generate accurate mass discrete and composite spectral libraries for metabolite identification and for SWATH mass spectrometry data processing. Discrete collision energy (5–100 eV) accurate mass spectra were collected for 532 metabolites from the human metabolome database (HMDB) by flow injection analysis and compiled into composite spectra over a large collision energy range (e.g., 10–70 eV). Full scan response factors were also calculated. Software tools based on accurate mass and predictive fragmentation were specially developed and found to be essential for construction and quality control of the spectral library. First, elemental compositions constrained by the elemental composition of the precursor ion were calculated for all fragments. Secondly, all possible fragments were generated from the compound structure and were filtered based on their elemental compositions. From the discrete spectra, it was possible to analyze the specific fragment form at each collision energy and it was found that a relatively large collision energy range (10–70 eV) gives informative MS/MS spectra for library searches. From the composite spectra, it was possible to characterize specific neutral losses as radical losses using in silico fragmentation. Radical losses (generating radical cations) were found to be more prominent than expected. From 532 metabolites, 489 provided a signal in positive mode [M+H]+ and 483 in negative mode [M-H]−. MS/MS spectra were obtained for 399 compounds in positive mode and for 462 in negative mode; 329 metabolites generated suitable spectra in both modes. Using the spectral library, LC retention time, response factors to analyze data-independent LC-SWATH-MS data allowed the identification of 39 (positive mode) and 72 (negative mode) metabolites in a plasma pool sample (total 92 metabolites) where 81 previously were reported in HMDB to be found in plasma.

RATIONALE:In peptide quantification by liquid chromatography/mass spectrometry (LC/MS), the optimization of multiple reaction monitoring (MRM) parameters is essential for sensitive detection. We have compared different approaches to build MRM assays, based either on flow injection analysis (FIA) of isotopically labelled peptides, or on the knowledge and the prediction of the best settings for MRM transitions and collision energies (CE). In this context, we introduce MRMOptimizer, an open-source software tool that processes spectra and assists the user in selecting transitions in the FIA workflow.METHODS:MS/MS spectral libraries with CE voltages from 10 to 70 V are automatically acquired in FIA mode for isotopically labelled peptides. Then MRMOptimizer determines the optimal MRM settings for each peptide. To assess the quantitative performance of our approach, 155 peptides, representing 84 proteins, were analysed by LC/MRM-MS and the peak areas were compared between: (A) the MRMOptimizer-based workflow, (B1) the SRMAtlas transitions set used 'as-is'; (B2) the same SRMAtlas set with CE parameters optimized by Skyline.RESULTS:51% of the three most intense transitions per peptide were shown to be common to both A and B1/B2 methods, and displayed similar sensitivity and peak area distributions. The peak areas obtained with MRMOptimizer for transitions sharing either the precursor ion charge state or the fragment ions with the SRMAtlas set at unique transitions were increased 1.8- to 2.3-fold. The gain in sensitivity using MRMOptimizer for transitions with different precursor ion charge state and fragment ions (8% of the total), reaches a ~ 11-fold increase.CONCLUSIONS:Isotopically labelled peptides can be used to optimize MRM transitions more efficiently in FIA than by searching databases. The MRMOptimizer software is MS independent and enables the post-acquisition selection of MRM parameters. Coefficients of variation for optimal CE values are lower than those obtained with the SRMAtlas approach (B2) and one additional peptide was detected. Copyright © 2017 John Wiley & Sons, Ltd.

In recent years, the biochemical study of lipids has transformed from slow multi-dimensional chromatographic separations and chemical derivatization strategies to higher throughput analysis by mass spectrometry. Advances in mass spectrometry have enabled in-depth lipidomic analyses with unparalleled qualitative and quantitative sensitivity. However, unambiguous identification and quantitation of lipid molecular species in total lipid extracts has proven to be difficult, primarily due to isobaric overlapping isobaric and isomeric species. There are greater than 100,000 lipid molecular species present in a typical biological lipid extract that occupy a narrow mass range (~400-1100 amu), making such overlap a significant problem.

Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.

The analysis of lipids by mass spectrometry (MS) can provide in-depth characterization for many forms of biological samples. However, such workflows can also be hampered by challenges like low chromatographic resolution for lipid separations and the convolution of mass spectra from isomeric and isobaric species. To address these issues, we describe the use of differential mobility spectrometry (DMS) as a rapid and predictable separation technique within a shotgun lipidomics workflow, with a special focus on phospholipids (PLs). These analytes, ionized by electrospray ionization (ESI), are filtered using DMS prior to MS analysis. The observed separation (measured in terms of DMS compensation voltage) is affected by several factors, including the m/z of the lipid ion, the structure of an individual ion, and the presence of chemical modifiers in the DMS cell. Such DMS separations can simplify the analysis of complex extracts in a robust and reproducible manner, independent of utilized MS instrumentation. The predictable separation achieved with DMS can facilitate correct lipid assignments among many isobaric and isomeric species independent of the resolution settings of the MS analysis. This leads to highly comprehensive and quantitative lipidomic outputs through rapid profiling analyses, such as Q1 and MRM scans. The ultimate benefit of the DMS separation in this unique shotgun lipidomics workflow is its ability to separate many isobaric and isomeric lipids that by standard shotgun lipidomics workflows are difficult to assess precisely, for example, ether and diacyl species and phosphatidylcholine (PC) and sphingomyelin (SM) lipids.

Purpose.: To quantify the molecular lipid composition of patient-matched tear and meibum samples and compare tear and meibum lipid molecular profiles. Methods.: Lipids were extracted from tears and meibum by bi-phasic methods using 10:3 tert-butyl methyl ether:methanol, washed with aqueous ammonium acetate, and analyzed by chip-based nanoelectrospray ionization tandem mass spectrometry. Targeted precursor ion and neutral loss scans identified individual molecular lipids and quantification was obtained by comparison to internal standards in each lipid class. Results.: Two hundred and thirty-six lipid species were identified and quantified from nine lipid classes comprised of cholesterol esters, wax esters, (O-acyl)-ω-hydroxy fatty acids, triacylglycerols, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine. With the exception of phospholipids, lipid molecular profiles were strikingly similar between tears and meibum. Conclusions.: Comparisons between tears and meibum indicate that meibum is likely to supply the majority of lipids in the tear film lipid layer. However, the observed higher mole ratio of phospholipid in tears shows that analysis of meibum alone does not provide a complete understanding of the tear film lipid composition.

Shotgun lipidomics has evolved into a myriad of multi-dimensional strategies for molecular lipid characterization, including bioinformatics tools for mass spectrum interpretation and quantitative measurements to study systems-lipidomics in complex biological extracts. Taking advantage of spectral mass accuracy, scan speed and sensitivity of improved quadrupole linked time-of-flight mass analyzers, we developed a bias-free global lipid profiling acquisition technique of sequential precursor ion fragmentation called MS/MSALL. This generic information-independent tandem mass spectrometry (MS) technique consists of a Q1 stepped mass isolation window through a set mass range in small increments, fragmenting and recording all product ions and neutral losses. Through the accurate MS and MS/MS information, the molecular lipid species are resolved, including distinction of isobaric and isomeric species, and composed into more precise lipidomic outputs. The method demonstrates good reproducibility and at least 3 orders of dynamic quantification range for isomeric ceramides in human plasma. More than 400 molecular lipids in human plasma were uncovered and quantified in less than 12 min, including acquisitions in both positive and negative polarity modes. We anticipate that the performance of sequential precursor ion fragmentation both in quality and throughput will lead to the uncovering of new avenues throughout the biomedical research community, enhance biomarker discovery and provide novel information target discovery programs as it will prospectively shed new insight into affected metabolic and signaling pathways.