Anal. Chem. 2006, 78, 3062-3071
Characterizing the Phospholipid Profiles in Mammalian Tissues by MALDI FTMS Jeffrey J. Jones, Sabine Borgmann, and Charles L. Wilkins*
Department of Chemistry and Biochemistry, University of Arkansas, University of Arkansas, Fayetteville, Arkansas 72701 Richard M. O’Brien
Department of Molecular Physiology & Biophysics, Vanderbilt University, Vanderbilt University Medical School, Nashville, Tennessee 37232
Discussed here is an analytical method for profiling lipids and phospholipids directly from mammalian tissues excised from Mus musculus (house mouse). Biochemical analysis was accomplished through the use of matrixassisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry, where whole tissue sections of mouse brain, heart, and liver were investigated. Lipid and phospholipid ions create complex MALDI mass spectra containing multiple ions with different m/z values corresponding to the same fundamental chemical species. When a computational sorting approach is used to group these ions, the standard deviation for observed relative chemical abundance can be reduced to 6.02%. Relative standard deviations of 10% are commonly accepted for standard chromatographic phospholipid analyses. Average mass measurement accuracy for 232 spectra representing three tissue types from 12 specimens was calculated to be 0.0053 Da. Further it is observed, that the data and the analysis between all the animals have nearidentical phospholipid contents in their brain, heart, and liver tissues, respectively. In addition to the need to accurately measure relative abundances of phospholipid species, it is essential to have adequate mass resolution for complete and accurate overall analysis. It is reasonable to make mass composition assignments with spectral resolving power greater than 8000. However, results from the present study reveal 14 instances (C12 carbon isotope) of multiple m/z ions having the same nominal value that require greater resolution in order that overlap will not occur. Spectra measured here have an average resolving power of 12 000. It is established that high mass resolution and mass accuracy coupled with MALDI ionization provide for rapid and accurate phospholipid analysis of mammalian tissue sections. Fourier transform mass spectrometry (FTMS) is becoming a widespread tool in biological analysis, finding its way as a resource in the “omic” sciences1 and bacterial taxonomy.2 Matrix-assisted laser desorption/ionization (MALDI)3,4 and electrospray ionization * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Tyers, M.; Mann, M. Nature 2003, 422, 193-197.
3062 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006
(ESI) mass spectrometry have contributed significantly to understanding biological systems.5 Microorganisms are routinely investigated by MALDI time-of-flight (TOF) mass spectrometry and high-resolution MALDI Fourier transform mass spectrometry for proteomicsandlowmolecularweightphospholipidcharacterization.6-8 FTMS provides exceptional resolution and accuracy over TOF and quadrupole ion trap mass spectrometers,9 with typical accurate mass accuracies of 10 ppm or better. The ionization efficiencies for phospholipids depend primarily on the hydrophilic headgroups that distinguish each of the lipids and, therefore, are more difficult to quantify.10 Phospholipids comprise the great majority of components observed in MALDI FTMS experiments when low m/z ions from whole cells and tissues are sampled.7,8,11 Lipids and phospholipids are significant components of all biological cell membranes and are a class of molecules that are greater in copy number than proteins and genetic sequence material as cell membrane components.12 Lipids are a family of biomolecules that play prominent roles in many critical metabolic and biochemical processes such as energy production and storage, the formation and functioning of cellular membranes, and signal transduction.13 Lipid analysis, referred to as lipidomics,14 aims at understanding the implications of lipid diversity and regulation that affects cellular functions. Lipid and phospholipid compositions can change dramatically as a result of environment and cellular activity, with the consequence that (2) Anhalt, J. P.; Fenselau, C. Anal. Chem. 1975, 47, 219-225. (3) Lay, J. O. Mass Spectrom. Rev. 2001, 20, 172-194. (4) Fenselau, C.; Demirev, P. A. Mass Spectrom. Rev. 2001, 20, 157-171. (5) van Baar, B. L. M. FEMS Microbiol. Rev. 2000, 24, 193-219. (6) Stump, M. J.; Jones, J. J.; Fleming, R. C.; Lay, J. O.; Wilkins, C. L. J. Am. Soc. Mass Spectrom. 2003, 14, 1306-1314. (7) Jones, J. J.; Stump, M. J.; Fleming, R. C.; Lay, J. O.; Wilkins, C. L. Anal. Chem. 2003, 75, 1340-1347. (8) Jones, J. J.; Stump, M. J.; Fleming, R. C.; Lay, J. O.; Wilkins, C. L. J. Am. Soc. Mass Spectrom. 2004, 15, 1665-1674. (9) Sleno, L.; Volmer, D. A.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2005, 16, 183-198. (10) Marto, J. A.; White, F. M.; Seldomridge, S.; Marshall, A. G. Anal. Chem. 1995, 67, 3979-3984. (11) Ho, Y. P.; Fenselau, C. Anal. Chem. 1998, 70, 4890-4895. (12) Lechevalier, M. P. Crit. Rev. Microbiol. 1977, 5, 109-210. (13) Lee, S. H.; Williams, M. V.; DuBois, R. N.; Blair, I. A. Rapid Commun. Mass Spectrom. 2003, 17, 2168-2176. (14) Kishimoto, K.; Urade, R.; Ogawa, T.; Moriyama, T. Biochem. Biophys. Res. Commun. 2001, 281, 657-662. 10.1021/ac0600858 CCC: $33.50
© 2006 American Chemical Society Published on Web 03/01/2006
Table 1. Survey about Current Analytical Techniques for the Determination of Phospholipids (PLs) in Biological Samples method for detecting PLs from whole cells and tissues
principle method of analysis
advantages
disadvantages
ref
fluorescence-based phospholipase assays
continuous fluorometric monitoring of enzymatic activity using self-quenching or excimer-forming probes
continuous PL detection
use of modified PLs necessary; FA structure not realized; time-consuming
42
detection of free fatty acid formation with, e.g., ADIFAB reagent (Molecular Probes) or choline with phospholipase assays
use of a fluorescent probe labeled to a rat intestinal FA binding protein with high binding affinity to FA
continuous PL detection
indirect assay due to detection of free FAs; FA chain length not realized; time-consuming
43, 44
anti-PL antibodies
immunocytochemical location of PLs in cells and tissues
localization of PLs to cell compartments
time-consuming; not quantitative
45
use of labeled PLs (radioactive or fluorescent)
determination of rates for PLs metabolism (flux, turnover rates, half-life of PLs)
imaging in vivo and in vitro possible; real-time approach
radioactive materials
46
thin-layer chromatography (TLC)
stationary phase on a glass plate; detection with spray reagents,47 colorimetric determination of phosphorus
inexpensive; fast; frequently applied standard technique; often used as first step of the analysis; high reproducibility; quantification performed by densitometry or image analysis
time-consuming; laborintensive organic solvent extraction and separation; standards are required; low resolution (determination of FA composition of a given PL is difficult)
19, 48, 49
high-performance liquid chromatography (HPLC)
detection with UV (typically, at 203-214 nm), light scattering,50 refractive index (RI) or flame ionization (FI)51
standard techniques; applicable to a preparative HPLC; often used as first step of the analysis
time-consuming; empirical selection of an appropriate solvent system; labor-intensive organic solvent extraction and separation; detection of PLs is the most challenging part; standards are required; low selectivity
19, 52-56]
fatty acid methyl ester (FAME) gas chromatography (GC) coupled with mass spectrometry (GC/MS)
separation of PLs using a carrier gas as mobile phase; detection often performed with MS; indirect detection after derivatizing PLs57
most important standard routine approach; highthroughput screening compatible
only free FAs detectable; derivatization57 is needed (might lead to rearrangements of the FA chains); time-consuming due to several hours of manual derivatization prior to GC analysis
58, 59
capillary electrophoresis (CE)
separate of ionic PLs by their size-to-charge ratio and frictional force within a small capillary filled with an electrolyte; detection with UV, UV-vis
high efficiency, relatively high resolution; small amounts of sample and solvent
solubilization of PLs; PLs have weak UV absorption; ambiguity in peak assignment; coupling to other detection methods such as MS is challenging due to the high voltage required for CE and the electrolyte composition; standards required
60, 61
micellar electrokinetic capillary chromatography (MEKC)
separation of PLs by differential partitioning between a pseudostationary micellar phase and an aqueous mobile phase within a microfluidic chip device followed by fluorescence detection
simple and efficient; small amounts of sample and solvent; HTS option (80 min per 384-well-plate)
PLs have weak UV absorption; ambiguity in peak assignment; standards required
62-65
detection of all compounds within a sample; 1H NMR can be applied in vivo detection of only PLs; quantitative; high selectivity
low sensitivity (13C); headgroup not realized
29
FA not realized; timeconsuming; susceptible to diamagnetic and paramagnetic polyvalent cations which are able to coordinate with the phosphodiesterfunctional group
29, 66, 67
nuclear magnetic resonance (NMR)
1H/13C
identification of PLs by measuring the chemical shifts of the PLs nuclei introduced by their different electron densities
31P
NMR
NMR
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Table 1 (Continued) method for detecting PLs from whole cells and tissues
principle method of analysis
advantages
disadvantages
ref
fast atom bombardment (FAB) MS
TOF MS
detection of parent ions along with fragmented ions; tandem MS enables structural characterization of PLs
moderate sensitivity; inherent present matrix background signals; relative high level of fragmentation
23-25, 68-71
pyrolysis coupled with GC/MS
bench-top quadrapole MS
GC is used to separate volatile lipids; quadrapole MS detection is routine
indirect characterization of lipids by analyzing chemical derivatives produced during the analysis
22, 59, 72, 73
electrospray ionization (ESI) MS
ESI FT MS
high resolving power for ions below m/z 1000; mass assignment accuracy
