Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

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.

Fast Electron Detachment Dissociation of Oligonucleotides in Electron-Nitrogen Plasma Stored in Magneto Radio-Frequency Ion Traps.

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.

Hybrid SWATH/MS and HR-SRM/MS acquisition for phospholipidomics using QUAL/QUANT data processing.

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. Graphical abstract.

Metabolomic spectral libraries for data-independent SWATH liquid chromatography mass spectrometry acquisition.

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. Graphical abstract Library generation workflow for LC-SWATH MS, using collision energy spread, accurate mass, and fragment annotation.

Optimization by infusion of multiple reaction monitoring transitions for sensitive quantification of peptides by liquid chromatography/mass spectrometry.

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.

Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry.

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.

Differential mobility spectrometry-driven shotgun lipidomics.

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.

Quantitative profiling of PE, MMPE, DMPE, and PC lipid species by multiple precursor ion scanning: a tool for monitoring PE metabolism.

We report a method for the simultaneous identification and quantification of phosphatidylethanolamine (PE), monomethyl-phosphatidylethanolamine (MMPE), dimethyl-phosphatidylethanolamine (DMPE), and phosphatidylcholine (PC) species in lipid extracts. The method employs a specific "mass-tag" strategy where DMPE, MMPE, and PE species are chemically methylated with deuterated methyliodide (CD(3)I) to produce PC molecules having class-specific mass offsets of 3, 6 and 9Da, respectively. The derivatized aminoglycerophospholipids release characteristic phosphorylcholine-like fragment ions having specific mass offsets that powers sensitive and quantitative analysis by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer. Using the mass-tag strategy, we could for the first time determine the stoichiometric relationship between the biosynthetic intermediates MMPE and DMPE, and abundant PE and PC species in a single mass spectrometric analysis. We demonstrated the efficacy of the methodology by conducting a series of biochemical experiments using stable isotope labeled ethanolamine to survey the activities and substrate specificities of enzymes involved in PE metabolism in Saccharomyces cerevisiae. Finally, we benchmarked the mass-tag strategy by specific and sensitive profiling of intermediate MMPE and DMPE species in liver.

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry.

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided approximately 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.

Dissociation of Biomolecules by an Intense Low-Energy Electron Beam in a High Sensitivity Time-of-Flight Mass Spectrometer.

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.

Software-aided detection and structural characterization of cyclic peptide metabolites in biological matrix by high-resolution mass spectrometry.

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.

Rapid screening and characterization of drug metabolites using multiple ion monitoring dependent product ion scan and postacquisition data mining on a hybrid triple quadrupole-linear ion trap mass spectrometer.

Multiple ion monitoring (MIM)-dependent acquisition with a triple quadrupole-linear ion trap mass spectrometer (Q-trap) was previously developed for drug metabolite profiling. In the analysis, multiple predicted metabolite ions are monitored in both Q1 and Q3 regardless of their fragmentations. The collision energy in Q2 is set to a low value to minimize fragmentation. Once an expected metabolite is detected by MIM, enhanced product ion (EPI) spectral acquisition of the metabolite is triggered. To analyze in vitro metabolites, MIM-EPI retains the sensitivity and selectivity similar to that of multiple reaction monitoring (MRM)-EPI in the analysis of in vitro metabolites. Here we present an improved approach utilizing MIM-EPI for data acquisition and multiple data mining techniques for detection of metabolite ions and recovery of their MS/MS spectra. The postacquisition data processing tools included extracted ion chromatographic analysis, product ion filtering and neutral loss filtering. The effectiveness of this approach was evaluated by analyzing oxidative metabolites of indinavir and glutathione (GSH) conjugates of clozapine and 4-ethylphenol in liver microsome incubations. Results showed that the MIM-EPI-based data mining approach allowed for comprehensive detection of metabolites based on predicted protonated molecules, product ions or neutral losses without predetermination of the parent drug MS/MS spectra. Additionally, it enabled metabolite detection and MS/MS acquisition in a single injection. This approach is potentially useful in high-throughout screening of metabolic soft spots and reactive metabolites at the drug discovery stage.

Combining Charge-Switch Derivatization with Ozone-Induced Dissociation for Fatty Acid Analysis.

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.

Qualitative analysis and quantitative assessment of changes in neutral glycerol lipid molecular species within cells.

Triacylglycerols (TAGs) and diacylglycerols (DAGs) are present in cells as a complex mixture of molecular species that differ in the nature of the fatty acyl groups esterified to the glycerol backbone. In some cases, the molecular weights of these species are identical, confounding assignments of identity and quantity by molecular weight. Electrospray ionization results in the formation of [M+NH4]+ ions that can be collisionally activated to yield an abundant product ion corresponding to the loss of ammonia plus one of the fatty acyl groups as a free carboxylic acid. A method was developed using tandem mass spectrometry (MS) and neutral loss scanning to analyze the complex mixture of TAGs and DAGs present in cells and to quantitatively determine changes in TAGs and DAGs molecular species containing identical fatty acyl groups in an experimental series. Eighteen different deuterium-labeled internal standards were synthesized to serve to normalize the ion signal for each neutral loss scan. An example of the application of this method was in the quantitative analysis of TAG and DAG molecular species present in RAW 264.7 cells treated with a Toll-4 receptor ligand, Kdo2-lipid A, in a time course study.

Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry.

The yeast Saccharomyces cerevisiae synthesizes three classes of sphingolipids: inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramides (MIPCs), and mannosyl-diinositolphosphoceramides (M(IP)2C). Tandem mass spectrometry of their molecular anions on a hybrid quadrupole time-of-flight (QqTOF) instrument produced fragments of inositol-containing head groups, which were specific for each lipid class. MS(n) analysis performed on a hybrid linear ion trap-orbitrap (LTQ Orbitrap) mass spectrometer with better than 3 ppm mass accuracy identified fragment ions specific for the amide-linked fatty acid and the long chain base moieties in individual molecular species. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends itself to rapid, comprehensive and structure-specific profiling of the molecular composition of sphingolipids and glycerophospholipids in important model organisms, such as fungi and plants.

A comparison of patient matched meibum and tear lipidomes.

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 by Sequential Precursor Ion Fragmentation on a Hybrid Quadrupole Time-of-Flight Mass Spectrometer.

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.

Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry.

Triacylglycerols (TAGs) are neutral lipids present in all mammalian cells as energy reserves, and diacylglycerols (DAGs) are present as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of TAGs and DAGs present in mammalian cells are quite complex, and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600 and 900 Da, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed, using tandem MS and neutral loss scanning, to quantitatively analyze changes in those glyceryl ester molecular species containing identical fatty acyl groups. This was carried out by neutral loss scanning of 18 common fatty acyl groups where the neutral loss corresponded to the free carboxylic acid plus NH(3). Deuterium-labeled internal standards were used to normalize the signal for each nominal [M+NH(4)](+) ion undergoing this neutral loss reaction. This method was applied in studies of TAGs in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo(2)-lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24-h time course after Kdo(2)-lipid A exposure, whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.

Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning.

We report a method for the identification and quantification of glycerophospholipid molecular species that is based on the simultaneous automated acquisition and processing of 41 precursor ion spectra, specific for acyl anions of common fatty acids moieties and several lipid class-specific fragment ions. Absolute quantification of identified species was linear within a concentration range of 10 nM-100 microM and was achieved by spiking into total lipid extracts a set of synthetic lipid standards with diheptadecanoyl (17:0/17:0) fatty acid moieties, representing six common classes of glycerophospholipids. The automated analysis of total lipid extracts was powered by a robotic nanoflow ion source and produced currently the most detailed description of the glycerophospholipidome.