Subscriber access provided by UNIV OF NEW ENGLAND
Article
Dual emitter nano-Electrospray Ionization coupled to Differential Ion Mobility Spectrometry-Mass Spectrometry for Shotgun Lipidomics James E Keating, and Gary L Glish Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01528 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 15, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Dual emitter nano-Electrospray Ionization coupled to Differential Ion Mobility Spectrometry-Mass Spectrometry for Shotgun Lipidomics James E. Keating and Gary L. Glish* Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA ABSTRACT: Current lipidomics workflows are centered around acquisition of large data sets followed by lengthy data processing. A dual nESI-DIMS-MS platform was developed to perform real-time relative quantification between samples, providing data required for biomarker discovery and validation more quickly than traditional ESI-MS approaches. Nanosprayer activity and DIMS compensation field settings were controlled by a LabVIEW program synced to the accumulation portion of the ion trap scan function, allowing for full integration of the platform with a commercial mass spectrometer. By comparing samples with short electrospray pulses rather than constant electrospray the DIMS and MS performance is normalized within an experiment, as signals are compared between individual mass spectra (ms time scale) rather than individual experiments (min-hr time scale). The platform was validated with lipid standards and extracts from nitrogen-deprived microalgae. Dual nESI-DIMS requires minimal system modification and is compatible with all traditional ion activation techniques and mass analyzers, making it a versatile improvement to shotgun lipidomics workflows.
Mass spectrometry provides high sensitivity and selectivity that can be applied to profile and quantify lipids in biologically relevant matrices. As a class of biomolecules, the structure of lipids is far more variable than those of nucleic acids or proteins. This provides unique challenges in their analysis, which has generally hindered the progress of lipidomics relative to proteomics or genomics.1 The diverse biological activity of lipids makes them useful biomarkers for a range of diseases as well as prime targets for therapeutic intervention. Differential expression of lipids is often used as an indicator of disease states, with examples including increased expression of triglycerides and cholesteryl esters in cardiovascular disease2 and aberrant phospholipid expression in multiple cancers.3 Lipid metabolism is also known to be involved in Alzheimer’s, Parkinson’s, and Niemann-Pick diseases.4 Direct infusion, or shotgun, lipidomics methods provide a means of identifying differential lipid expression in diseased samples, but requires that a control sample is analyzed concomitantly, or that an instrument response has been previously determined for a given condition. For novel applications, determining a threshold response for a condition is work-intensive when compared to the use of a control sample, especially when the large number of potential differentially expressed lipid species is considered. Control samples allows for direct comparisons of instrument response between control conditions and experimental conditions, whether experimental conditions include known or suspected disease states, environmental exposure, or therapeutic intervention. As the data of interest is a comparison of lipid expression between samples, an ionization source that allows for the real-time comparison of two or more samples simultaneously can increase throughput and account for biases inherent to performing separate analyses. The most commonly used ionization technique for the analysis of biomolecules is electrospray ionization (ESI). ESI
generates gas-phase ions directly from solution, and is easily coupled to mass spectrometers with an atmospheric pressure interface.5 A low-flow rate regime of electrospray ionization, nano-electrospray ionization (nESI), provides increased sensitivity, reduced sample consumption, and can be controlled electrokinetically.6,7 Electrokinetic control allows for pulsed operation, where multiple nano-ESI emitters are sampled separately.8,9 Multi-emitter ESI source designs have been used for a number of applications, most notably as a means of improving mass accuracy by using a reference sprayer with known compounds for internal mass calibration within an experiment.10-12 The ability to control nESI electrokinetically and the interest in comparing lipid expression between experimental conditions prompted the design and validation of the dual nESI source used in this work. A major limitation of lipidomics is sample complexity. Typical lipid samples are organic extracts of biological media, as the hydrophobicity of lipids allows for high extraction efficiencies of most lipid classes.13 These extracts can contain hundreds of unique lipids spanning a large range of concentrations. For example, a tissue sample extract is likely to contain very high concentrations of structurally-important lipids (e.g. phospholipid bilayer), and low concentrations of signaling lipids that may be important in describing a disease state. Presence of high concentration lipids can interfere with detection of low concentration species, which limits the ability to identify differential expression in these samples. Use of chromatographic separation techniques can alleviate the issue of complexity, but introduces problems including inconsistent ionization efficiency with the use of gradient elution, brief signal for individual lipid species during elution (on the order of seconds), and increased analysis time. Ion mobility spectrometry (IMS) techniques provide an alternative means of handling complex samples that introduces fewer interfering biases by separating lipid species post-ionization. The magnitude of ionization suppression does increase
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
without the use of a pre-ionization separation (chromatography), though it is constant during an experiment rather than dependent on time and chromatographic conditions (i.e. coeluting compounds). Ionization suppression can limit the ability to detect low abundance species, but the use of DIMS with a trapping-type mass spectrometer can offset this disadvantage by allowing for selective accumulation of target species after filtering by differential mobility.14 Separation of ions by their mobility in an electric field has been realized in a number of instrumental designs. The simplest of these is drift-tube ion mobility (DT-IMS), where ions are carried through a drift region by a uniform electric field and opposed by a flow of inert gas.15 This requires gating of ions into the drift region, reducing the duty cycle of the analysis. Ions separated by their collision cross sections are measured by their arrival time at the mass analyzer. DT-IMS has been previously used in the separation of complex lipid samples and in a discovery-based approach to lipid isomer identification.16,17 Newer variations on DT-IMS, including traveling wave ion mobility spectrometry (TWIMS) and trapped ion mobility spectrometry (TIMS), offer some advantages including lower voltage requirements and tunable resolution, but function on the same principle of separation by collision cross sections as DTIMS18,19. Another implementation, differential ion mobility spectrometry (DIMS) separates ions based on the difference between their high and low electric field mobility’s (KH and KL).20 In DIMS, an asymmetric waveform that alternates between high and low electric fields of opposite polarity is applied to a set of planar electrodes situated in front of the inlet to the mass spectrometer. The peak amplitude of the asymmetric waveform (V0P) is referred to as the dispersion voltage. A dc voltage, referred to as a compensation voltage is applied to one of the electrodes to adjust the trajectory of ions, allowing ions with a specific differential mobility to reach the mass spectrometer. A linear ramp of this voltage permits transmission of ions with successive differential mobilities. The width of the gap between the two planar electrodes is used to calculate the dispersion field (ED) and compensation field (EC) from the dispersion voltage and compensation voltage, respectively. The gas number density (N) is used to normalize the electric field (V/cm) for convenient conversion of a DIMS separation method between devices and experimental conditions (temperature, pressure, etc.). E/N (V cm2) values are expressed in Townsends (1 Td = 10-17 V cm2 and high-field ion mobility conditions begin at approximately 30 Td (7.47 kV/cm for operation at 1 atm and 25°C). As transmission occurs at a specific compensation field, DIMS offers the advantage of functioning as a constant-transmission filter, where separation conditions are held constant during analysis to improve selectivity for an analyte. DIMS also provides a more orthogonal separation to mass analysis than traditional IMS approaches, as the correlation between m/z and ΔK (|KH-KL|) is weaker than the correlation between m/z and KL.20 This is primarily because KL is only a function of an ion’s average collisional cross section whereas KH depends on a greater number of factors including ion polarity, ion-molecule interactions, and electric field strength21. This is particularly important for the separation of lipids, as a large number of isobaric and isomeric species occupy a relatively narrow range of m/z. As m/z and KL are closely related, separation of a given pair of isomeric lipids with traditional drift tube ion mobility would require long path lengths and separation time when compared to
the path length and time of a DIMS separation. The use of solvent modifiers adds additional flexibility in DIMS experimental design, as ion-molecule interactions with the carrier gas have a marked effect on the EC required to transmit an ion through the device.22–24 These advantages of DIMS over other ion-mobility techniques have led to a great deal of interest in the use of DIMS for lipid analysis.25–27 The constructed dual nESI source is readily coupled to DIMS-MS as the nanospray emitter activity and the DIMS EC control can be synced to the timing of the ion trap scan function. Though coupled to a quadrupole ion trap in these experiments, the dual nESI-DIMS platform is compatible with other mass analyzers, including triple quadrupole, time-of-flight, ion cyclotron resonance, and orbitrap designs. The lack of mechanical or fluid movement once the nanospray emitters are in place allows for a robust instrumental setup that is capable of rapid shotgun lipidomics experiments. Experimental Samples. A bovine heart extract and a synthetic lipid mixture (composition given in Supplementary Information) were purchased from Avanti Polar Lipids (Alabaster, AL). Balb C mouse plasma was purchased from Fisher Scientific (Hampton, NH). Ammonium acetate (ACS grade), methanol (Optima grade), dichloromethane (Optima grade), and isopropanol (ACS grade) were purchased from Fisher Scientific (Hampton, NH). Methyl tert-butyl ether (ACS Grade) was purchased from MilliporeSigma (St. Louis, MO). Chlamydomonas reinhardtii (CC2895 6145c mt- strain) samples: 2-300 mL seed cultures were started from solid Tris acetate phosphate (TAP) media C. reinhardtii plates and grown until early log phase. Cultures were grown under continuous light at 22°C under 100 µmol m–2 s–1 using white light (24 hr lighting) in 500 mL flasks on an Innova 2300 (New Brunswick Scientific, Enfield, CT, USA) shaker at 250 rpm using TAP medium. Samples were taken at log phase with cell concentrations of 2.0 × 107 cells/mL. To examine the effects of nitrogen deprivation on the C. reinhardtii lipid content, cultures were pelleted (5 min using 2000 x G) and one 300 mL culture was resuspended in nitrogen-replete media and the other culture was resuspended in fresh TAP media to serve as the control. After 24 hr. in nitrogen-replete media or fresh TAP, samples were immediately harvested using centrifugation (5 min, 2000 x G) and stored at -80 °C until use. Lipid extraction. Methyl-tert-butyl ether (MTBE) was used to extract lipids from C. reinhardtii cell pellets and mouse plasma according to literature.28 Briefly, 375 µL of methanol was added to 50 mg of cell pellets and vortexed for 30 s. 1.25 mL of MTBE was added and the solution was incubated for 10 minutes. 310 µL of water was added for phase separation and the solution was centrifuged for 5 min (15,600 x G). The MTBE layer was transferred to a new centrifuge tube and dried using a Thermo Scientific SpeedVac (SPD1010, 45 °C, 5.1 torr). Extracted lipids were reconstituted in 1 mL of 10 mM ammonium acetate in 50/50 (v/v) methanol/dichloromethane. Instrumentation. A dual-emitter nanoelectrospray ion source was constructed using two X-Y positioning stages, allowing for independent positioning of the two emitters. The dual nESI source and a custom-built planar DIMS device (10mm electrodes, 0.3 mm gap) were coupled to a Bruker HCT ion trap mass spectrometer. A Narishige micropipette puller was used to generate glass nanoelectrospray emitters and stainless-steel wires were placed inside each emitter to provide
ACS Paragon Plus Environment
Page 2 of 8
Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry electrical connectivity to the sample solution. EMCO C25N high voltage power supplies were used to provide voltage to each emitter separately. The dual-emitter ion source and DIMS were controlled using a National Instruments DAQ (PCI-6713) and a TTL output from the mass spectrometer synced to ion accumulation. 10 mM ammonium acetate in 50/50 (v/v) methanol/dichloromethane was used as electrospray solvent. DIMS Carrier Gas. The drying gas (N2 by default) of the Bruker HCT mass spectrometer was used as the DIMS carrier gas, and the flow rate and temperature were controlled by the Bruker EsquireControl software. The DIMS device is designed such that the drying gas flows around the housing and is pulled back through the electrodes based on the gas flow into the glass transfer capillary. The total flow rate through the DIMS electrodes is then dictated by the fore pressure of the mass spectrometer and the flow through the glass transfer capillary. Solvent modifiers were added to the carrier gas by intersecting the flow of drying gas with the flow from a syringe pump (ColeParmer, SK-74900-05) prior to the gas being heated. Solvent modifier concentrations were calculated by volume and assuming ideal gas behavior. Results and Discussion Design and Characterization of dual nESI Source. The dual nESI source was designed to allow for automatic or manual control of each nanospray emitter using the combination of inlet capillary voltage and external high voltage power supplies. In positive ionization mode, a negative voltage is applied to the inlet capillary (-2.5 kV), one of the external high voltage supplies is on (-2 kV), and the other external high voltage supply is held at ground potential. Electrospray is generated from the nanospray emitter coupled to the grounded external supply based on the electric field gradient between the solution in the emitter tip and the inlet capillary. The active external power supply (-2 kV) prevents electrospray from the other emitter, as the electric field gradient is not sufficient to initiate formation of a Taylor cone. In negative ionization mode, the inlet capillary is held at ground potential and the external power supplies provide the negative polarity high voltage for initiation of electrospray (-2.5 kV). The activity of the external power supplies is controlled by a TTL input (0-5 V) which is coupled to the ion trap scan function and controlled by LabVIEW. Emitters can be controlled manually by a LabVIEW toggle button, or automatically based on a selected number of ion accumulations (equivalent to spectral averaging). For the case of n = 1 ion accumulation, the external supplies are alternated after each ion accumulation (falling-edge triggered), providing extra time for stabilization of electrospray during the mass analysis portion of the scan function. A schematic representation of the dual nESI source and the associated timing diagram are given in Supplementary Figure S-1. Performance of the dual-emitter nanoelectrospray-DIMS-MS platform was characterized using a bovine heart extract and synthetic lipid mixture. In pulsed operation, the duration of each electrospray pulse dictates the length of an experiment, and faster pulses come at the expense of signal reproducibility. The relative standard deviation (RSD) associated with pulsing after each ion accumulation (equivalent to a 1 spectral average, approximately 200 ms) is significantly higher for all measured lipids than the RSD associated with pulsing after 20 ion accumulations (equivalent to 20 spectral averages, approximately 4s). These results are illustrated in the form of a box plot of extracted ion intensities and associated error measurements from
Figure 1. Box plots of extracted ion intensities for individual lipid species in a bovine heart extract and synthetic lipid mixture for electrospray pulse durations equivalent to 1 (yellow), 5 (grey), 10 (orange), and 20 (blue) spectral averages.
five replicate pulses in Figure 1. Lipid annotations are consistent with the LIPID MAPS classification system.29,30 Tabulated RSD values are given in Supplementary Table S-1. Ultimately, the pulse time used in an experiment will be dictated by the needs of a given application. For example, a pulse time equivalent to 1 spectral average may be suitable for rapid qualitative or screening applications, and longer pulse times more appropriate for quantitative experiments. The trade-off between speed and reproducibility is not unique to the pulsing nanospray setup and is expected based on the relationship between measurement uncertainty and number of measurements. Though not a unique phenomenon, characterization of pulse times provides a fundamental understanding of both system performance and the length of time required to complete an experiment. Different concentrations of bovine heart extract in electrospray solvent with a constant concentration of synthetic lipids (internal standard) were used to simulate differential expression. Qualitative stability and reproducibility of pulsing of the two samples are demonstrated by the total ion chronogram and two extracted ion chronograms of triglyceride (TG) species
Figure 2. Alternating signal from 1 and 4 µg/mL bovine heart extract with a synthetic lipid mixture as internal standard shown as the total ion chronogram (Blue), extracted ion chronogram of TG (52:2) (Green), and extracted ion chronogram of TG (54:3) (Gold).
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(TG (52:2) from bovine heart extract and TG (54:3) from the synthetic mixture) in Figure 2. The inclusion of lipid class specific internal standards in each emitter corrects for differences in absolute signal intensity from individual pulses and for minor differences in sprayer positioning. Accurate absolute quantitation of lipid species using ESIMS is known to require a large number of internal standards within a lipid class to account for differences in ionization efficiency inherent to changes in the lipid acyl chain lengths.31 Comparing relative abundance of lipids between control and stressed conditions circumvents this requirement, as lipids are being compared only to the identical species in the other sprayer. For the experiments presented here, the synthetic lipid mixture provides a suitable internal standard for relative and semi-quantitative applications. The use of additional internal standards is compatible with the platform presented here and would enable absolute quantification within the same experiment as the comparison between samples. DIMS Separation coupled to dual nESI Source. DIMS is implemented easily into the dual nESI-MS experimental design, as the post-ionization separation is independent of sample conditions (i.e. no special mobile phase considerations) and the compensation field, EC, can be controlled by the same LabVIEW program as the dual nESI source. The DIMS device has two main modes of operations, EC scanning, where ions over a range of differential mobilities are successively transmitted to the mass spectrometer, and static EC filtering, where only ions of a specific differential mobility are transmitted to the mass spectrometer. The scanning mode is ideal for untargeted experiments, as well as for method development for targeted analysis. Static filtering requires that the EC for transmission of a specific ion is known but can be used for rapid targeted analyses with no limitation on the number of discrete EC ‘steps’ that can be taken for multi-compound analysis. Separation of lipid classes by DIMS has previously been shown with the use of isopropanol as a solvent modifier in the DIMS carrier gas.22 The separation of lipid classes in a synthetic lipid mixture using isopropanol in the DIMS carrier gas is shown in Supplementary Figure S-2. Lipid class separation by DIMS functions analogously to separation of lipid classes by normal phase liquid chromatography (NPLC) (including hydrophilic interaction liquid ion chromatography), though the spacedispersive DIMS separation can be scanned through in as little as 2 minutes, and a time-dispersive NPLC separation requires upwards of 30 minutes.32 Separation of lipids (in the form of an EC scan) in a bovine heart extract using isopropanol as a solvent modifier and mass spectra collected at specific EC values are shown in Figure 3. A comparison between the mass spectrum without the use of DIMS and the mass spectrum of EC = 332 V/cm (Figure 3A and Figure 3Ciii) demonstrates significant improvement in the signal-to-noise (S/N) for low abundance sphingomyelin species (e.g. m/z 732 (SM(d36:1)) and m/z 802, SM(d41:1)), as well as simplification of the mass spectra that aids in assignment of peaks to unique lipid species. Beyond S/N improvements and spectral simplification with the use of DIMS, the EC required to transmit a lipid ion can be used as a confirmation of lipid class. For example, glycerophosphocholines are often identified based on detection of the m/z 184 phosphocholine headgroup during tandem mass spectrometry (MS/MS), but the choline ion is observable in the MS/MS of sphingomyelin species as well. For this case, the EC required
for transmission provides another dimension of lipid class confirmation, functioning analogously to retention time matching
Figure 3. A) Mass spectrum of bovine heart extract in 10mM ammonium acetate 50/50 methanol/dichloromethane with inset of an expanded view of m/z 700-820 to show low abundance sphingolipid peaks. B) EC scan of bovine heart extract in 10mM ammonium acetate 50/50 methanol/dichloromethane. C) Mass spectra of bovine heart extract in 10mM ammonium acetate 50/50 methanol/dichloromethane at static EC values for transmission of triglycerides, glycerophosphocholines, and sphingolipids at EC = 156, 286, and 332 V/cm, respectively.
in chromatography. Simulated Differential Expression. Differential expression of lipid species was simulated by preparing solutions of a bovine heart extract at different concentrations and a synthetic lipid mixture at the same concentration. Fold changes were measured between a 1 and 2 µg/mL (total extracted mass) bovine heart extract using a 50 ng/mL (each lipid) synthetic lipid mixture as an internal standard. Nanospray emitters were loaded with 20 µL of one of the lipid solutions and placed in the dual nESI source. In this design, all of the lipids in the bovine heart extract are ‘differentially expressed’ by a factor equivalent to the difference in sample dilution. Performance of the source was then evaluated by comparing the measured fold changes to known fold changes. The dual nESI-DIMS-MS platform was setup to measure the simulated differential expression by collecting full scan mass spectra at static EC values previously determined to transmit specific lipid classes from each nanospray emitter consecutively. Using unmodified carrier gas (100% N2), triglycerides and glycerophosphocholine/glycerophosphoethanolamines were measured at EC values of 260 V/cm and 350 V/cm, respectively. Measured abundances of TG, PC, and PE species from each emitter were normalized using TG (54:3), PC (28:2), and PE (28:2) from the synthetic lipid mixture. Normalized abundances were used to calculate measured fold changes and compared to the known fold change from sample preparation. Measured fold changes for individual lipid species at each of the EC values and error bars denoting the deviation in measurement for alternating electrospray between the two nanospray emitters (n=5, 12 sec each) are given in Figure 4. The fold changes for the majority of the measured lipid species were accurately measured with 20% bias (5 of 31 total). Most of the measurements with >20% bias were of low abundance
ACS Paragon Plus Environment
Page 4 of 8
Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry TG species (TG (48:0), TG (51:3), TG (51:2), and TG (53:2)) that were difficult to measure in the more dilute of the two samples, so the excessively high fold change measurements can be attributed to overcoming limitations of signal intensity when measuring these TG’s in the less concentrated sample. Excluding measurements of the low abundance TG lipids, the average fold change measured for the TG, PE, and PC lipids was 2.06 ± 0.19, suggesting little to no systematic bias. Measurement of simulated differential expression in a mouse plasma lipid extract (2.23x fold change from dilution) by pulsing the nanospray emitters during each step of a EC scan, rather than by holding EC constant, had an average fold change of 2.36 ± 0.32 (Supplementary Figure S-3).
Figure 5. A) Extracted ion chronograms of glycerophosphoethanolamine and glycerophosphocholine isobars in a bovine heart extract separated using 0.25% isopropanol in nitrogen carrier gas. B) Measured differential expression in full scan mass spectra between a 1 and 4 µg/mL bovine heart extract at the EC values for transmission of the respective isobars. Figure 4. Fold changes measured between 1 and 2 µg/mL bovine heart extract solutions by alternating electrospray between two nanospray emitters (n=5, 12s per pulse) at static EC values chosen for selective transmission of triglyceride and glycerophosphocholine/glycerophosphoethanolamine species (EC = 260 V/cm and EC = 350 V/cm). TG (54:3), PC (28:2), and PE (28:2) were used as internal standards. TG species denoted by orange markers were measured with low intensity in the 1 µg/mL sample (4%-11% of base peak intensity).
As discussed previously, distinguishing isobars of different lipids classes is readily achieved with the use of solvent modifiers in the DIMS carrier gas. Protonated glycerophosphocholine and glycerophosphoethanolamine isobars were separated using 0.25% isopropanol in nitrogen as a carrier gas, and the EC values for transmission of each isobar were used to measure differential expression between a 1 and 4 µg/mL (total extracted mass) bovine heart extract using a 50 ng/mL (each lipid) synthetic lipid mixture as an internal standard. Negative mode MS/MS data supporting the proposed lipid identifications is given in Supplementary Figure S-4 and Supplementary Table S-2. The separation of protonated PE and PC isobars and measured differential expression are shown in Figure 5. Isobaric pairs are resolved with R = 2.13 for m/z 768.6 and R = 1.31 for m/z 782.6, with protonated PE species transmitting at EC = 100 V/cm and protonated PC species transmitting at EC = 250 V/cm (ED = 190 Td, carrier gas of 0.25% isopropanol in N2) DIMS-MS/MS experiments reveal a sodiated PC ([PC (34:1) + Na] + co-transmitting with the protonated PE at m/z 782.6 under the dispersion field and modifier conditions used for full scan measurement of differential expression (Figure 6). The fold changes were measured with
