Easy, Fast, and Reproducible Quantification of Cholesterol and Other Lipids in Human Plasma by Combined High Resolution MSX and FTMS Analysis
- 806 Downloads
Reliable, cost-effective, and gold-standard absolute quantification of non-esterified cholesterol in human plasma is of paramount importance in clinical lipidomics and for the monitoring of metabolic health. Here, we compared the performance of three mass spectrometric approaches available for direct detection and quantification of cholesterol in extracts of human plasma. These approaches are high resolution full scan Fourier transform mass spectrometry (FTMS) analysis, parallel reaction monitoring (PRM), and novel multiplexed MS/MS (MSX) technology, where fragments from selected precursor ions are detected simultaneously. Evaluating the performance of these approaches in terms of dynamic quantification range, linearity, and analytical precision showed that the MSX-based approach is superior to that of the FTMS and PRM-based approaches. To further show the efficacy of this approach, we devised a simple routine for extensive plasma lipidome characterization using only 8 μL of plasma, using a new commercially available ready-to-spike-in mixture with 14 synthetic lipid standards, and executing a single 6 min sample injection with combined MSX analysis for cholesterol quantification and FTMS analysis for quantification of sterol esters, glycerolipids, glycerophospholipids, and sphingolipids. Using this simple routine afforded reproducible and absolute quantification of 200 lipid species encompassing 13 lipid classes in human plasma samples. Notably, the analysis time of this procedure can be shortened for high throughput-oriented clinical lipidomics studies or extended with more advanced MSALL technology (Almeida R. et al., J. Am. Soc. Mass Spectrom. 26, 133–148 ) to support in-depth structural elucidation of lipid molecules.
KeywordsCholesterol Multiplexing Shotgun lipidomics High resolution mass spectrometry Orbitrap
Shotgun lipidomics platforms using nanoelectrospray ionization and high resolution mass spectrometry (MS) technology afford comprehensive lipidome analysis with confident identification and accurate quantification of several hundred lipid molecules in a single sample [1, 2, 3, 4, 5, 6]. Applications of this technology have prompted mechanistic insights into the regulation of lipid metabolism [2, 4, 7, 8], membrane-related processes [9, 10, 11, 12], and lipid–protein interactions [13, 14]. In addition, the technology has also pinpointed reliable lipid biomarkers of cardiovascular disease [15, 16]. Notably, a prerequisite for global lipidome analysis is that each sample should be analyzed by a series of mass spectrometric routines executed in both positive and negative ion mode (e.g., MSALL ). However, in this analytical setting it is challenging to quantify non-esterified cholesterol (cholestenol) since it ionizes poorly by (nano)electrospray ionization and undergoes in-source fragmentation to produce a positively charged cholestadiene fragment ion.
Several solutions are available for circumventing this issue and enabling gold-standard absolute quantification of cholesterol (e.g., pmol/μL plasma) by shotgun lipidomics. One option is to improve the ionization efficiency of cholesterol, and an appropriate internal standard such as 2H7-cholesterol, by using chemical derivatization with either sulfur trioxide or acetyl chloride to produce sulfate or acetate derivatives, respectively [17, 18]. Notably, these approaches entail extra sample handling steps, and an additional sample injection and mass spectrometric analysis. An alternative and simpler approach is to use high resolution Fourier transform (FT) MS analysis on Orbitrap-based machines and monitor the cholesterol-derived in-source cholestadiene fragment ions with m/z 369.3516 and m/z 376.3955 released from endogenous cholesterol and the internal standard 2H7-cholesterol, respectively . The latter approach, however, is not specific for non-esterified cholesterol and can prompt inaccurate quantification since cholesteryl esters (CEs) also undergo in-source fragmentation to produce the cholestadiene fragment ion with m/z 369.3516 (Supplementary Figure S1).
With the recent development of more sensitive hybrid quadrupole-Orbitrap-based instruments (i.e., Orbitrap Fusion and Q-Exactive) it is now possible to use high resolution full scan FTMS analysis to monitor cholesterol and 2H7-cholesterol as intact ammonium adducts (e.g., [cholesterol+NH4]+, m/z 404.3887), detected as low intensity ions but with high signal-to-noise ratios (Supplementary Figure S1) . Moreover, it is also possible to quantify cholesterol levels using parallel reaction monitoring (PRM) of ammoniated cholesterol and 2H7-cholesterol, and thereby consecutively monitor the intensity of cholestadiene and 2H7-cholestadiene fragment ions, respectively, in two separate FTMS2 (Fourier transform tandem mass spectrometry) scans . Furthermore, a third approach is to monitor cholesterol and 2H7-cholesterol using novel multiplexed MS/MS (MSX) technology [20, 21, 22, 23, 24]. In this approach, selected precursor ions are sequentially (1) isolated by a quadrupole mass analyzer, (2) fragmented in a collision cell, and (3) fragment ions from each of the selected precursor ions are trapped inside the collision cell prior to being routed together to an Orbitrap mass analyzer for simultaneous detection. Notably, at the present time there has been no effort to compare the performance of the three approaches available for direct cholesterol quantification on hybrid quadrupole-Orbitrap-based instruments.
Here, we report on the analytical merits of absolute quantification of cholesterol in human plasma using full scan FTMS, PRM, and MSX analysis. To this end, we optimized the performance of the three approaches and assessed their individual performances in terms of dynamic quantification range and analytical precision. This assessment demonstrated that both MSX and PRM analysis outperforms full scan FTMS in terms of dynamic range and linearity. Moreover, we also found that MSX-based cholesterol quantification is more precise compared with PRM and FTMS-based analysis. To demonstrate the efficacy of the MSX method, we devised a simple shotgun lipidomics routine for simultaneous and absolute quantification of cholesterol and other lipid molecules in human plasma. This routine uses an easy-to-use, commercially available internal standard mixture (SPLASH Lipidomix) that is spiked into human plasma, a single lipid extraction step and 6 min of combined high resolution FTMS and MSX analysis. Using this routine we were able to reproducibly quantify the absolute levels of 200 lipid species from 13 lipid classes in a single injection of a human plasma extract.
Materials and Methods
Chemicals and Lipid Standards
Chloroform, methanol, and 2-propanol were purchased from Rathburn Chemicals (Walkerburn, Scotland). Ammonium acetate and ammonium formate were from Sigma-Aldrich (Buchs, Switzerland). All solvents and chemicals were HPLC grade. SPLASH Lipidomix (containing 213 μM PC 15:0-18:1-2H7, 8.0 μM PE 15:0-18:1-2H7, 5.4 μM PS 15:0-18:1-2H7, 38.1 μM PG 15:0-18:1-2H7, 10.7 μM PI 15:0-18:1-2H7, 10.7 μM PA 15:0-18:1-2H7, 48.2 μM LPC 18:1-2H7, 10.9 μM LPE 18:1-2H7, 541 μM CE 18:1-2H7, 5.5 μM MAG 18:1-2H7, 16.0 μM DAG 15:0-18:1-2H7, 70.5 μM TAG 15:0-18:1-2H7-15:0, 41.9 μM SM 18:1-2H9, and 254 μM (25, 26, 26, 26, 27, 27, 27-2H7)-cholesterol) and (25, 26, 26, 26, 27, 27, 27-2H7)-cholesterol were from Avanti Polar Lipids (Alabaster, AL, USA). β-Sitosterol was from Sigma-Aldrich (Brøndby, Denmark).
Human blood samples were collected from five healthy volunteers (three males; two females). Informed consent was obtained from all individuals before participation. The study was approved by The Regional Scientific Ethical Committees for Southern Denmark and performed in accordance with the Helsinki Declaration. The volunteers (body mass index 23.3–30.5 kg/m2; 25–40 years of age) fasted overnight. Venous blood samples were collected into 4 mL K2 EDTA vacutainer tubes. EDTA plasma was separated by centrifugation (2500 g, 5 min, 4 °C), immediately snap-frozen in liquid nitrogen, and stored at –80 °C until further analyses.
Animal experiments were conducted in accordance with the Danish law on Animal Experiments (LBK no. 1306 - 23/11/2007, amendments § 1 nr. 612 - 14/06/2011) and approved by the Danish Animal Experiment Inspectorate. C57BL/6J mice of 12 wk of age were fasted for 2 h and subsequently anesthetized. Blood sampling was performed by cardiac puncture into K3 EDTA micro tubes (Sarstedt, Nümbrecht, Germany). EDTA plasma was separated by centrifugation (3000 g, 15 min, 4 °C), immediately snap-frozen in liquid nitrogen, and stored at –80 °C until further processing.
Human and mouse plasma samples (8 μL) were subjected to lipid extraction at 4 °C as previously described [3, 25]. Briefly, plasma aliquots were diluted with 155 mM ammonium formate to a final volume of 200 μL and spiked with 12 μL of SPLASH Lipidomix. Subsequently samples were extracted with 990 μL of chloroform/methanol (10:1, v/v) and mixed for 120 min at 1400 rpm. After 3 min centrifugation at 1500 g, the lower organic phase was collected and vacuum evaporated.
Mass Spectrometric Analysis
Lipid extracts and synthetic lipid standards were dissolved in chloroform/methanol/2-propanol (1:2:4 v/v/v) containing 7.5 mM ammonium acetate and loaded in 96-well plates (Eppendorf, Hamburg, Germany). Samples (10 μL) were infused with the robotic nanoflow ion source TriVersa NanoMate (Advion Biosciences, Ithaca, NY, USA) using nanoelectrospray chips (flow rate of 200 nL/min) and analyzed in positive ion mode using an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, San Jose, CA, USA). The instrument was tuned and calibrated approximately every 2 wk following manufacturer’s recommended procedures. Ionization voltage was +0.96 kV and back pressure was 1.25 psi. The temperature of the ion transfer tube was 275 °C. S-lens radio frequency (rf) level was set to 60%. Positive ion mode MS analysis of the lipid extracts was performed by (1) high resolution FTMS analysis of the m/z range 345–605, (2) high resolution FTMS of the m/z range 470–1200, (3) MSX analysis of both m/z 404.4 ([cholesterol +NH4]+) and m/z 411.4 ([2H7-cholesterol +NH4]+) (Supplementary Figure S3), (4) PRM analysis of m/z 404.4 ([cholesterol +NH4]+), and (5) PRM analysis of m/z 411.4 ([2H7-cholesterol +NH4]+). Each sample was analyzed for 6 min. All full scan FTMS data were acquired in profile mode, using a max injection time of 100 ms, automated gain control for an ion target of 105, three microscans, and a target resolution setting of 500,000. FTMS2 data were collected in profile mode within the range m/z 340–440 with the following settings: higher-energy collisional dissociation (HCD) fragmentation using normalized collision energy to 8%, maximum injection time of 600 ms, automated gain control for an ion target of 5 × 104, five microscans, a target resolution of 30,000, and a quadrupole ion isolation window of 1.5 u.
Lipid Identification and Quantification
Lipid species detected by high resolution FTMS, PRM, and MSX analysis with a mass accuracy less than 5 ppm were identified and quantified using ALEX software and SAS 9.3 [1, 26]. Lipid species were quantified by normalizing their intensity to the intensity of an internal lipid standard of identical lipid class and multiplying by the spike amount of the internal lipid standard . Only lipid species detected in all technical replicates and present in all subjects (n = 5) are reported.
Results and Discussion
Monitoring of Cholesterol by MSX Analysis
Full scan FTMS analysis (Figure 1a) demonstrated that (1) cholesterol and 2H7-cholesterol can be detected as ammonium adducts, but with a very low intensity compared with other ions (Figure 1a and Supplementary Figure S1C), (2) the intensity ratio between ammoniated cholesterol and 2H7-cholesterol was 2.8:1, and that (3) in-source fragmentation produces a highly abundant cholestadiene fragment ion (m/z 369.3516) and a low abundant 2H7-cholestadiene fragment ion (m/z 376.3955). The higher intensity of m/z 369.3516 corroborates the notion that CEs undergo in-source fragmentation (Supplementary Figure S1). The PRM analysis showed that (1) fragmentation of ammoniated cholesterol yields the cholestadiene fragment ion with m/z 369.3516 (Figure 1b), (2) fragmentation of ammoniated 2H7-cholesterol yields the 2H7-cholestadiene fragment ion with m/z 376.3955 (Figure 1c), and that (3) the intensity ratio between these fragment ions was 2.6:1. Furthermore, the MSX analysis showed that the cholesterol- and 2H7-cholesterol-derived fragment ions with m/z 369.3516 and m/z 376.3955 could be detected simultaneously in a single detection event (Figure 1d), having an intensity ratio of 2.6:1. These results highlight that it is possible, on a hybrid quadrupole-Orbitrap mass spectrometer, to directly detect cholesterol and 2H7-cholesterol using three different approaches and that these produce similar results. However, this simple comparison does not by itself demonstrate which approach is the most precise, accurate, and sensitive for routine applications.
Optimizing MSX-Based Cholesterol Quantification
To try to minimize the in-source fragmentation of cholesterol and 2H7-cholesterol, and thereby potentially improve the detection of intact ammoniated sterol molecules by FTMS, PRM, and MSX analysis, we also tested the influence of the instrument’s ion entrance setting. This analysis showed that using an ion entrance S-lens radio frequency value of 60% was optimal for detection of intact ammoniated sterol molecules by full scan FTMS analysis (Supplementary Figure S2A) and also for the detection of cholestadiene-based fragment ions by FTMS2-based analysis (Supplementary Figure S2B).
Dynamic Quantification Range of MSX Analysis
MSX Analysis Improves Analytical Precision
To further assess the performance of the three approaches, we determined their analytical precision for quantification of cholesterol in human and mouse plasma, two sample matrices having different concentrations of non-esterified cholesterol. To this end, we spiked a human plasma sample with 2H7-cholesterol and subjected this sample to lipid extraction followed by six repeated mass spectrometric analyses with recording of full scan FTMS, PRM, and MSX data. This experiment was repeated on three different days to assess interday precision.
Intraday and Interday Precision of Cholesterol Quantification in Human Plasma Samples
Day 1 (n=6)
Day 2 (n=6)
Day 3 (n=6)
μM ± SD
μM ± SD
μM ± SD
μM ± SD
999 ± 10
874 ± 7
1032 ± 12
968 ± 71
988 ± 17
884 ± 25
1022 ± 28
965 ± 64
1078 ± 78
946 ± 59
1174 ± 136
1066 ± 132
Quantification of Cholesterol in Human Plasma by MSX Analysis
Profiling the Plasma Lipidome by High Resolution FTMS and MSX Analysis
Here, we evaluated the use of novel MSX technology for gold-standard absolute quantification (e.g., μM or mg/dL) of non-esterified cholesterol in human plasma samples. We demonstrate that the performance of the MSX-based approach is comparable to PRM-based analysis in terms of accuracy, linearity, and dynamic quantification range, and better in terms of analytical precision. Moreover, we also show that cholesterol quantification by both MSX- and PRM-based analysis is superior to that of full scan FTMS analysis. Although these shotgun lipidomics-based approaches, as well as techniques based on chemical derivatization [17, 18], provide easy and fast analytical strategies for quantification of cholesterol, they all have the same limitation, which is the assumption that the intact precursor ion with m/z 404.3887 and the fragment ion with m/z 369.3516 are derived from only one cholestenol isomer, namely cholest-5-en-3-ol (i.e., cholesterol). As such, users of these approaches should be aware that the accuracy of the cholesterol quantification can potentially be hampered by the presence of other cholestenol isomers, such as cholest-7-en-3-ol (i.e., lathosterol). This, however, should only be of concern when studying cohorts of patients or cells having severe defects in de novo sterol biosynthesis. In such scenarios it could be worthwhile to explore whether the MS3 fragmentation capabilities of the herein used instrumentation (Orbitrap Fusion) could enable specific detection of isomeric cholestenols. Alternatively, quantification of cholesterol (and other sterols) can also be done (with higher sensitivity) by combing chemical derivatization, reversed-phase chromatography, and selected ion monitoring on a triple quadrupole mass spectrometer . However, compared with the 6 min MSX-based analysis, this approach entails additional sample preparation (~1 h for derivatization and solvent evaporation) and a ~40 min chromatography run per sample (plus additional time for column reconditioning and blank runs to clean the column).
To demonstrate the efficacy of the MSX-based cholesterol quantification, we devised a simple routine that capitalizes on using only a few microliters of plasma that is spiked with a novel, commercially available ready-to-use internal standard mixture (SPLASH Lipidomix) and a single sample injection that affords cholesterol quantification by MSX analysis and quantification of sterol esters, glycerolipids, glycerophospholipids, and sphingolipids by full scan FTMS analysis. A key advantage of using the MSX-based method is that it does not require extra sample preparation steps with chemical derivatization to enhance ionization efficiency of non-esterified cholesterol and that it can be easily integrated with a palette of other acquisition procedures available on hybrid quadrupole-Orbitrap-based mass spectrometers (e.g., MSALL ). As such, we note that the analysis time of this routine can be shortened to ~2 min for high throughput-oriented analysis or alternatively be combined with MSALL technology with an analysis time of ~20 min to allow in-depth structural characterization of molecular lipid species (e.g., decipher the composition of fatty acyl moieties in detected lipid molecules). Finally, we note that the MSX-based approach is also applicable for quantifying cholesterol levels in tissue biopsies .
The authors thank Karina Vejrum Sørensen (Department of Endocrinology, Odense University Hospital) for human plasma samples, and Ditte Neess, and Nils J. Færgeman (Department of Biochemistry and Molecular Biology, University of Southern Denmark) for mouse plasma samples. The authors thank members of the Ejsing laboratory for advice and helpful discussions and Elena Sokol for expert advice on Orbitrap Fusion operation. This work was supported by the Danish Council for Strategic Research (11-116196), the University of Southern Denmark (SDU2020), and the VILLUM Center for Bioanalytical Sciences (VKR023179).
- 5.Sales, S., Graessler, J., Ciucci, S., Al-Atrib, R., Vihervaara, T., Schuhmann, K., Kauhanen, D., Sysi-Aho, M., Bornstein, S.R., Bickle, M., Cannistraci, C.V., Ekroos, K., Shevchenko, A.: Gender, contraceptives, and individual metabolic predisposition shape a healthy plasma lipidome. Sci. Rep. 6, 27710 (2016)CrossRefGoogle Scholar
- 6.Surma, M.A., Herzog, R., Vasilj, A., Klose, C., Christinat, N., Morin-Rivron, D., Simons, K., Masoodi, M., Sampaio, J.L.: An automated shotgun lipidomics platform for high throughput, comprehensive, and quantitative analysis of blood plasma intact lipids. Eur. J. Lipid Sci. Technol. 117, 1540–1549 (2015)CrossRefGoogle Scholar
- 8.Surma, M.A., Klose, C., Peng, D., Shales, M., Mrejen, C., Stefanko, A., Braberg, H., Gordon, D.E., Vorkel, D., Ejsing, C.S., Farese Jr., R., Simons, K., Krogan, N.J., Ernst, R.: A lipid E-MAP identifies Ubx2 as a critical regulator of lipid saturation and lipid bilayer stress. Mol. Cell. 51, 519–530 (2013)CrossRefGoogle Scholar
- 9.Klemm, R.W., Ejsing, C.S., Surma, M.A., Kaiser, H.J., Gerl, M.J., Sampaio, J.L., de Robillard, Q., Ferguson, C., Proszynski, T.J., Shevchenko, A., Simons, K.: Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network. J. Cell. Biol. 185, 601–612 (2009)CrossRefGoogle Scholar
- 11.Osman, C., Haag, M., Potting, C., Rodenfels, J., Dip, P.V., Wieland, F.T., Brügger, B., Westermann, B., Langer, T.: The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria. J. Cell. Biol. 184, 583–596 (2009)CrossRefGoogle Scholar
- 15.Tarasov, K., Ekroos, K., Suoniemi, M., Kauhanen, D., Sylvanne, T., Hurme, R., Gouni-Berthold, I., Berthold, H.K., Kleber, M.E., Laaksonen, R., Marz, W.: Molecular lipids identify cardiovascular risk and are efficiently lowered by simvastatin and PCSK9 deficiency. J. Clin. Endocrinol. Metabol. 99, E45–E52 (2014)CrossRefGoogle Scholar
- 16.Cheng, J.M., Suoniemi, M., Kardys, I., Vihervaara, T., de Boer, S.P., Akkerhuis, K.M., Sysi-Aho, M., Ekroos, K., Garcia-Garcia, H.M., Oemrawsingh, R.M., Regar, E., Koenig, W., Serruys, P.W., van Geuns, R.J., Boersma, E., Laaksonen, R.: Plasma concentrations of molecular lipid species in relation to coronary plaque characteristics and cardiovascular outcome: Results of the ATHEROREMO-IVUS study. Atherosclerosis. 243, 560–566 (2015)CrossRefGoogle Scholar
- 17.Sandhoff, R., Brügger, B., Jeckel, D., Lehmann, W.D., Wieland, F.T.: Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry. J. Lipid Res. 40, 126–132 (1999)Google Scholar
- 18.Liebisch, G., Binder, M., Schifferer, R., Langmann, T., Schulz, B., Schmitz, G.: High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Biochim. Biophys. Acta (BBA) – Mol. Cell Biol. Lipids. 1761, 121–128 (2006)CrossRefGoogle Scholar
- 21.Michalski, A., Damoc, E., Hauschild, J.P., Lange, O., Wieghaus, A., Makarov, A., Nagaraj, N., Cox, J., Mann, M., Horning, S.: Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol. Cell. Proteom. 10, M111.011015 (2011)CrossRefGoogle Scholar
- 28.Quehenberger, O., Armando, A.M., Brown, A.H., Milne, S.B., Myers, D.S., Merrill, A.H., Bandyopadhyay, S., Jones, K.N., Kelly, S., Shaner, R.L., Sullards, C.M., Wang, E., Murphy, R.C., Barkley, R.M., Leiker, T.J., Raetz, C.R.H., Guan, Z., Laird, G.M., Six, D.A., Russell, D.W., McDonald, J.G., Subramaniam, S., Fahy, E., Dennis, E.A.: Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 51, 3299–3305 (2010)CrossRefGoogle Scholar