A simple desalting method for direct MALDI mass spectrometry profiling of tissue lipids

Direct MALDI-mass spectrometry (MALDI-MS) profiling of tissue lipids often observes isobaric phosphatidylcholine (PC) species caused by the endogenous alkali metal ions that bias the relative abundance of tissue lipids. Fresh rat brain cryosections were washed with 70% etha­nol (EtOH), water (H2O), or 150 mM ammonium acetate (NH4Ac), and the desalting effectiveness of each fluid was evaluated by MALDI-MS profiling of PC and sphingomyelin (SM) species in tissue and in the washing runoff. The results indicated that EtOH and H2O only partially desalted the tissue lipids, yet both substantially displaced the tissue lipids to the washing runoffs. On the other hand, NH4Ac effectively desalted the tissue lipids and produced a runoff containing no detectable PCs or SMs. NH4Ac wash also unveiled the underlying changes of PCs and SMs in the infarcted rat cortex previously masked by edema-caused increase of tissue sodium. The MS/MS of an isobaric PC in the infarcted cortex revealed the precursor change as the result of NH4Ac wash and confirmed the desalting effectiveness of such wash. Other than desalting, NH4Ac wash also removes contaminants in tissue, enhances the overall spectral quality, and benefits additionally in profiling of biological molecules in tissue. Direct MALDI-mass spectrometry (MALDI-MS) profiling of tissue lipids often observes isobaric phosphatidylcholine (PC) species caused by the endogenous alkali metal ions that bias the relative abundance of tissue lipids. Fresh rat brain cryosections were washed with 70% etha­nol (EtOH), water (H2O), or 150 mM ammonium acetate (NH4Ac), and the desalting effectiveness of each fluid was evaluated by MALDI-MS profiling of PC and sphingomyelin (SM) species in tissue and in the washing runoff. The results indicated that EtOH and H2O only partially desalted the tissue lipids, yet both substantially displaced the tissue lipids to the washing runoffs. On the other hand, NH4Ac effectively desalted the tissue lipids and produced a runoff containing no detectable PCs or SMs. NH4Ac wash also unveiled the underlying changes of PCs and SMs in the infarcted rat cortex previously masked by edema-caused increase of tissue sodium. The MS/MS of an isobaric PC in the infarcted cortex revealed the precursor change as the result of NH4Ac wash and confirmed the desalting effectiveness of such wash. Other than desalting, NH4Ac wash also removes contaminants in tissue, enhances the overall spectral quality, and benefits additionally in profiling of biological molecules in tissue. Alkali metal ions such as potassium and sodium are essential constituents in all biological organisms. A slight change in the concentration of these ions will significantly impact the organisms in osmotic pressure and fluid volume regulation, threshold maintenance of the excitable membranes, and in other aspects of homeostasis. However, these alkali metal ions usually become the inevitable constituent of, and frequently the interference to, the mass spectrometry analysis of biological samples. Previous MS studies of biological lipids reported that phosphatidylcholines (PCs) were preferentially ionized and detected in their alkalinated forms (1Jackson S.N. Wang H.Y. Woods A.S. In situ structural characterization of phosphatidylcholines in brain tissue using MALDI-MS/MS.J. Am. Soc. Mass Spectrom. 2005; 16: 2052-2056Crossref PubMed Scopus (156) Google Scholar, 2Han X. Gross R.W. Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2001; 295: 88-100Crossref PubMed Scopus (295) Google Scholar, 3Sugiura Y. Konishi Y. Zaima N. Kajihara S. Nakanishi H. Taguchi R. Setou M. Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry.J. Lipid Res. 2009; 50: 1776-1788Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 4Hsu F.F. Turk J. Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisional-activated dissociation on a triple stage quadrupole instrument.J. Am. Soc. Mass Spectrom. 2001; 12: 61-79Crossref PubMed Scopus (106) Google Scholar). Therefore, the highly abundant endogenous potassium and sodium ions might have conceivably biased the mass spectrometry presentations of tissue PCs (3Sugiura Y. Konishi Y. Zaima N. Kajihara S. Nakanishi H. Taguchi R. Setou M. Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry.J. Lipid Res. 2009; 50: 1776-1788Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Changes in tissue cations due to the pathological effects such as edema (5Gotoh O. Asano T. Koide T. Takakura K. Ischemic brain edema following occlusion of the middle cerebral artery in the rat. I: The time courses of the brain water, sodium and potassium contents and blood-brain barrier permeability to 125I-albumin.Stroke. 1985; 16: 101-109Crossref PubMed Scopus (272) Google Scholar, 6Slivka A. Murphy E. Horrocks L. Cerebral edema after temporary and permanent middle cerebral artery occlusion in the rat.Stroke. 1995; 26: 1061-1065, discussion 1065–1066Crossref PubMed Google Scholar) further enhanced the ionization of alkalinated PCs and SMs in the direct MALDI profiling (7Wang H.Y. Liu C.B. Wu H.W. Kuo J.S. Direct profiling of phospholipids and lysophospholipids in rat brain sections after ischemic stroke.Rapid Commun. Mass Spectrom. 2010; 24: 2057-2064Crossref PubMed Scopus (47) Google Scholar). Adding lithium (1Jackson S.N. Wang H.Y. Woods A.S. In situ structural characterization of phosphatidylcholines in brain tissue using MALDI-MS/MS.J. Am. Soc. Mass Spectrom. 2005; 16: 2052-2056Crossref PubMed Scopus (156) Google Scholar), potassium (3Sugiura Y. Konishi Y. Zaima N. Kajihara S. Nakanishi H. Taguchi R. Setou M. Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry.J. Lipid Res. 2009; 50: 1776-1788Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), or cesium (7Wang H.Y. Liu C.B. Wu H.W. Kuo J.S. Direct profiling of phospholipids and lysophospholipids in rat brain sections after ischemic stroke.Rapid Commun. Mass Spectrom. 2010; 24: 2057-2064Crossref PubMed Scopus (47) Google Scholar, 8Schiller J. Süß R. Petković M. Hilbert N. Müller M. Zschörnig O. Arnhold J. Arnold K. CsCl as an auxiliary reagent for the analysis of phosphatidylcholine mixtures by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).Chem. Phys. Lipids.. 2001; 113: 123-131Crossref PubMed Scopus (56) Google Scholar) in the MALDI matrix solution was attempted to circumvent this obstacle. However, protonated PCs and SMs were still easily detected with their alkalinated counterparts in most of the MALDI spectra acquired by this approach. Therefore, the profile of tissue lipids so revealed was still a biased pattern. Desalting samples prior to subjecting them for instrument analysis has long been a standard procedure in sample processing for biological MS studies. Direct MALDI-MS profiling and imaging of tissue proteins and peptides generally adopt an ethanol wash step to remove the endogenous salts and other interference in tissue before spectrometrically analyzing the tissue sections (9Goodwin R.J. Pennington S.R. Pitt A.R. Protein and peptides in pictures: imaging with MALDI mass spectrometry.Proteomics. 2008; 8: 3785-3800Crossref PubMed Scopus (128) Google Scholar, 10MacAleese L. Stauber J. Heeren R.M. Perspectives for imaging mass spectrometry in the proteomics landscape.Proteomics. 2009; 9: 819-834Crossref PubMed Scopus (60) Google Scholar). However, it remains to be determined if this ethanol wash is suitable for tissue lipid studies. In this study, we evaluated the desalting effectiveness and the overall suitability of ethanol wash for tissue lipids. We also explored the possibility of desalting tissue lipids with water based on the general immiscibility between water and lipids. Meanwhile, the desalting effectiveness of ammonium acetate (NH4Ac) solution isotonic to the mammalian physiological milieu was investigated. The profiles of PCs and SMs in tissue section and in the washing runoff were used together in the evaluation of each desalting fluid. It was reasoned that a suitable desalting fluid should effectively remove the alkali metal ions bound to tissue lipids while minimally disturbing or displacing the tissue lipids to the washing runoff. The most suitable desalting fluid was further tested on infarcted rat brain sections where the edema-induced increase of tissue sodium masked the revelation of underlying changes of PCs and SMs by MALDI-MS. Comparison of the MALDI-MS/MS result of an isobaric PC in the infarcted cortex revealed the change of precursor by NH4Ac wash and confirmed the desalting effectiveness of the chosen desalting fluid. MALDI matrix 2,6-dihydroxyacetophenone (DHA) was purchased from Fluka (Bauch, Switzerland). Ammonium acetate (ACS grade) was purchased from Sigma-Aldrich (St. Louis, MO). All the organic solvents were acquired from J. T. Baker or Mallinckrodt (Mallinckrodt Baker Inc., Phillipsburg, NJ). All the solvents were of HPLC grade except ethanol, which was 99.98%. Animal care and use were in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. The experimental procedures were approved by the Institutional Animal Care and Use Committee of National Sun Yat-Sen University before the study began. Male Sprague-Dawley rats (280–350g) were euthanized with isoflurane and decapitated immediately after the spontaneous breathing had ceased. The brains were rapidly dissected from the cranium and snap-frozen in isopentane prechilled on dry ice. The frozen brains were quickly wrapped in aluminum foil and stored at −80°C until needed (11Wang H.Y. Jackson S.N. McEuen J. Woods A.S. Localization and analyses of small drug molecules in rat brain tissue sections.Anal. Chem. 2005; 77: 6682-6686Crossref PubMed Scopus (70) Google Scholar). Surgical induction of ischemic stroke in rats, the neurological evaluation of the animals, and histological verification have been described previously (7Wang H.Y. Liu C.B. Wu H.W. Kuo J.S. Direct profiling of phospholipids and lysophospholipids in rat brain sections after ischemic stroke.Rapid Commun. Mass Spectrom. 2010; 24: 2057-2064Crossref PubMed Scopus (47) Google Scholar). Rat brains were collected 24 h after stroke induction. The frozen rat brains were cut into 14 µm coronal sections with a cryostat (Leica CM 3050, Nossloch, Germany) and collected on the glass slides coated with indium-tin oxide (ITO slides; 7-10 Ω, 2.5 cm × 7.5 cm) for mass spectrometry profiling. All the coronal sections were cut and collected serially from the region approximately 1.2 ± 0.2 mm rostral to Bregma (12Paxinos G. Watson C. The Rat Brain in Stereotaxic Coordinates.Compact 3rd. ed. Academic Press, Inc., San Diego, CA1997Google Scholar) along the anterior-posterior axis. After a very brief thaw-mounting, the collected sections were immediately placed in a desiccator evacuated by a vacuum pump (RV5, Edwards, West Sussex, UK) for 15 min. Seventy percent (v/v) ethanol (EtOH), water (H2O; 18.2 MΩ/cm, purified by Synergy Ultrapure Water System, Millipore Co.), and 150 mM NH4Ac solution (pH = 6.9) (13Berman E.S. Fortson S.L. Checchi K.D. Wu L. Felton J.S. Wu K.J. Kulp K.S. Preparation of single cells for imaging/profiling mass spectrometry.J. Am. Soc. Mass Spectrom. 2008; 19: 1230-1236Crossref PubMed Scopus (58) Google Scholar) were selected as the desalting fluids for this desalting fluid was tested on brain brain section was with of desalting fluid. section the the tissue section was at to a and the desalting fluid was to the tissue of the washing fluid was to a and of tissue section by the desalting fluid. The washing runoff was collected for the lipid and of runoff be collected from the washing of tissue Care was to of tissue section and the by the of washing fluid. The washing was usually in min. After the tissue section was immediately in the vacuum for to the washing fluid and the cortex region was with of solution in 70% for MALDI-MS and MALDI-MS/MS in the collected washing runoff were with the of and J. M. A for the and of from Chem. Full Text PDF PubMed Google Scholar). the of lipid the organic was to a glass sample and the was a of The was in of of and = with volume of of this was on the for MALDI-MS analysis of lipids in the extracts of the washing runoffs. The analyses of PC and in tissue and in the desalting were on a MALDI mass with a laser The instrument was for both MALDI-MS profiling and MALDI-MS/MS MALDI-MS ions from laser were collected and into MALDI-MS/MS ions from laser for precursor and each were collected and into The collected spectra were with The was a was of lipids that of the E. S. Murphy M. T. G. of the for Lipid Res. 2009; 50: Full Text Full Text PDF PubMed Scopus Google Scholar). the desalting effectiveness of these we the profiles of PC and in the rat cortex with from sections washed with and a MALDI-MS profile of PCs and SMs in rat cortex that is by the PC and However, the protonated and species are easily as the rat brain PC and species by MALDI-MS (7Wang H.Y. Liu C.B. Wu H.W. Kuo J.S. Direct profiling of phospholipids and lysophospholipids in rat brain sections after ischemic stroke.Rapid Commun. Mass Spectrom. 2010; 24: 2057-2064Crossref PubMed Scopus (47) Google Scholar). We have a isobaric PC all the the PC and profile in rat cortex after EtOH wash. It that the relative of the PC and species after EtOH wash. the tissue PC was as abundant as PC after EtOH wash. and and counterparts and were by EtOH wash as a be the result of of tissue sodium and the of tissue PC and or the by EtOH wash. EtOH wash the and PCs in tissue, this remove the sodium and potassium bound to tissue of the phosphatidylcholine sphingomyelin and other small molecular species in rat brain section by MALDI-MS (7Wang H.Y. Liu C.B. Wu H.W. Kuo J.S. Direct profiling of phospholipids and lysophospholipids in rat brain sections after ischemic stroke.Rapid Commun. Mass Spectrom. 2010; 24: 2057-2064Crossref PubMed Scopus (47) Google or or PC or PC in a profile of PC and species in rat cortex after desalting wash. The PC and species in rat cortex after the brain section was washed with 70% ethanol The PC and species in rat cortex after the brain section was washed with water (H2O; 18.2 The PC and species in rat cortex after the brain section was washed with 150 mM ammonium acetate = We tested H2O is an desalting fluid for PC and in tissue. the profile of PC and after the brain section was washed with H2O where a of and PCs was However, to the result of EtOH H2O wash remove the alkali metal ions in tissue. In after H2O the protonated and and were no detectable in tissue. of NH4Ac as the desalting fluid for tissue lipids was following the a MS profile of PC and in rat cortex after NH4Ac wash. The was by the protonated PC PC and PC at and their species were no of the PCs also after NH4Ac wash. the molecular ions at and that isobaric PC NH4Ac wash other highly abundant and PC these isobaric molecular species were the protonated PC species In to PC and we also detected the abundant in tissue after NH4Ac wash. Other than profiling the lipids in tissue, we also the PC and displaced to the runoff by the desalting the PC and in the EtOH washing runoff of the section for abundant PC and in their and forms in the yet of tissue lipids to the runoff by EtOH wash. result and the tissue lipid profile in that EtOH is as the desalting fluid for the direct profiling studies on tissue lipids. the PC and in the H2O washing runoff of the section for It that protonated PC species the spectral However, the collected runoff We were to the of such the most and wash with A brief of the collected runoff produced a and a small of at the of the It that were from the tissue section and to the in the runoff the tissue section after the in situ lipid profile that H2O is a tissue desalting abundant PC and in the runoff and the of tissue by H2O wash together have H2O as a suitable desalting fluid for the in situ lipid profiling studies. the lipids in the NH4Ac washing runoff of the section for It was that the overall spectral and the of the in were than in and It was due to the abundance the mass However, the no PC or species were in result and the tissue lipid profile in NH4Ac as the suitable desalting fluid that effectively removes the sodium and potassium bound to the tissue lipids yet or no of the in situ PC or the desalting of we the lipids in the infarcted rat brain section after it was washed with of a profile of lipids in the infarcted rat cortex before NH4Ac wash that is biased the PCs and (7Wang H.Y. Liu C.B. Wu H.W. Kuo J.S. Direct profiling of phospholipids and lysophospholipids in rat brain sections after ischemic stroke.Rapid Commun. Mass Spectrom. 2010; 24: 2057-2064Crossref PubMed Scopus (47) Google Scholar). It was to the underlying changes of PCs by this profile due to the that the of the for the PCs and the of the for the species were in the the lipid profile in the infarcted rat cortex from the section that was immediately to that of profile the increase in and that is from the interference of their alkalinated also the changes in the relative of PC PC PC and PC in rat of brain by ischemic stroke was previously with the of PCs in the brain R.M. X. Sun significantly phosphatidylcholine by and after Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R.M. is by and after cerebral in Res. PubMed Scopus (58) Google Scholar). Therefore, the profile of PCs in a of PCs in the cortex after The runoff analysis of the section for result in of PCs or the desalting effectiveness of NH4Ac we the MALDI-MS/MS analyses of an isobaric PC at from the infarcted cortex before and after the brain sections were washed with a MALDI-MS/MS result of the species before the section was washed with both the protonated PC and the PC be together as the precursor for the MS/MS Therefore, the MS/MS result is to the that the precursor as the The abundant an for the PC and the at and the at in all the that the precursor a PC R.M. of lipid species by MALDI mass spectrometry.J. Lipid Res. 2009; 50: Full Text Full Text PDF PubMed Scopus Google Scholar, of phosphatidylcholine phospholipids by mass Commun. Mass Spectrom. 2001; PubMed Scopus Google Scholar, F.F. Turk J. Structural determination of sphingomyelin by tandem mass spectrometry with electrospray Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar, F.F. Turk J. quadrupole mass studies on the Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar). The was generally as the of the protonated it also be from the PC in we were to in the at a of protonated and PC Other than the abundant between and were also MS/MS result the of the precursor in tissue before NH4Ac wash. on the other hand, a MS/MS result of the precursor from the infarcted cortex after the section was washed with The was by the the was it was in The the and the were no of the between and in were abundant ions that together a from that in the MS/MS profile in the precursor as a protonated PC and the of tissue desalting by NH4Ac wash. A desalting suitable for direct profiling of tissue lipids only effectively removes the alkali metal ions in tissue, it should also the isobaric species caused by these endogenous The use of such desalting should also minimally the tissue lipids and the of the tissue In this study, we have that NH4Ac solution isotonic to the general mammalian physiological is for the tissue desalting the desalting fluids The MS profile of the brain sections washed with this solution a profile of PCs and SMs of and of washing runoff no detectable of tissue PCs or SMs. desalting wash only the spectral profile of tissue PCs and it also the overall spectral and enhances the of abundant in the brain this desalting we are to the changes of brain PCs and SMs by ischemic stroke that have been by the changes of tissue potassium and sodium due to edema in the infarcted In mass spectrometry of salts NH4Ac in other salts used in the biological a concentration isotonic to the physiological of the sample that in this study, the ions in the solution a the tissue section and conceivably with the endogenous ions in general for their effects on the biological and the of the displaced endogenous ions by wash will the tissue alkali metal ions to the runoff. The ions be easily from tissue In all direct profiling of tissue lipids from sections washed with we have yet to PC or species in their that the overall and processing of tissue section is for the desalting the underlying lipid profile is the constituents of each PC species be further revealed by MALDI-MS/MS with containing alkali metal ions such as lithium or In to we have also evaluated the desalting effectiveness of ammonium used in the mass spectrometry brain sections washed with of 150 mM ammonium (pH = the lipid profiles in tissue section and in the runoff were to from sections desalted with Therefore, for the of tissue desalting, these ammonium salts Other than the washing used in this study, we have also tested washing to lipids in the rat brain The sections were in a containing NH4Ac for or were of each in the desalted the tissue lipids. However, the PC after washing the was or the of was the of tissue section was In of such we the desalting of the rat brain sections with washing in of tissue sample the sample processing used for the direct MS profiling or imaging of proteins and peptides in tissue, EtOH wash is generally a of desalting and a of for sections cut from the frozen tissue In to remove the lipid interference in tissue, sections are usually washed with solvents or the after the EtOH wash (9Goodwin R.J. Pennington S.R. Pitt A.R. Protein and peptides in pictures: imaging with MALDI mass spectrometry.Proteomics. 2008; 8: 3785-3800Crossref PubMed Scopus (128) Google Scholar, 10MacAleese L. Stauber J. Heeren R.M. Perspectives for imaging mass spectrometry in the proteomics landscape.Proteomics. 2009; 9: 819-834Crossref PubMed Scopus (60) Google Scholar, Stauber J. C. Heeren R.M. for tissue imaging by imaging 2009; 9: PubMed Scopus Google Scholar, R. M. A. R. M. MALDI-MS direct tissue analysis of using organic Chem. PubMed Scopus Google Scholar). to of EtOH is as a of lipid in most of the studies. it is the EtOH desalting results in this study with this However, based on this yet were in of desalting EtOH wash is an desalting for tissue studies further direct profiling studies of tissue PCs and SMs using MALDI in EtOH might have the lipid profiles of of (1Jackson S.N. Wang H.Y. Woods A.S. In situ structural characterization of phosphatidylcholines in brain tissue using MALDI-MS/MS.J. Am. Soc. Mass Spectrom. 2005; 16: 2052-2056Crossref PubMed Scopus (156) Google Scholar, S.N. Wang H.Y. Woods A.S. M. T. Direct tissue analysis of phospholipids in rat brain using and Am. Soc. Mass Spectrom. 2005; 16: PubMed Scopus Google Scholar, S.N. Wang H.Y. Woods A.S. Direct profiling of lipid distribution in brain tissue using Chem. 2005; 77: PubMed Scopus Google Scholar). the profiles of in situ PCs and SMs with their should be with sample processing that the of R.M. Murphy as a of matrix for mass Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar). H2O as a desalting fluid for tissue lipids than cells (13Berman E.S. Fortson S.L. Checchi K.D. Wu L. Felton J.S. Wu K.J. Kulp K.S. Preparation of single cells for imaging/profiling mass spectrometry.J. Am. Soc. Mass Spectrom. 2008; 19: 1230-1236Crossref PubMed Scopus (58) Google Scholar) and the tissue and results in of tissue The of tissue SMs after H2O wash is due to which is by than PCs in the studies A for by Lipid Res. 24: Full Text PDF PubMed Google Scholar, M. J. of by detected by electrospray ionization mass spectrometry.J. A. 2010; PubMed Scopus Google Scholar). Other than desalting tissue we have also tested the NH4Ac solution for the of contaminants such as or used it as a general to the for the in situ MS profiling. In both the NH4Ac solution results the sections were and We that this NH4Ac wash into sample processing for other of tissue profiling studies often the in the mass spectrometry Therefore, this washing conceivably further in studies and benefits in and other studies of In a yet to the tissue sections with NH4Ac solution that is suitable for direct mass spectrometry profiling of lipids in tissue has been in this of this desalting on tissue to pathological changes the effectiveness and of this The benefits in and by this will conceivably other mass spectrometry studies in and other of The of National Sun Yat-Sen for in instrument ethanol water indium-tin oxide ammonium acetate phosphatidylcholine