New Applications of Mass Spectrometry in Lipid Analysis

Abstract

Mass spectrometry has emerged as a powerful tool for the analysis of all lipids. Lipidomic analysis of biological systems
using various approaches is now possible with a quantitative measurement of hundreds of lipid molecular species. Although
availability of reference and internal standards lags behind the field, approaches using stable isotope-labeled derivative
tagging permit precise determination of specific phospholipids in an experimental series. The use of reactivity of ozone has
enabled assessment of double bond positions in fatty acyl groups even when species remain in complex lipid mixtures. Rapid
scanning tandem mass spectrometers are capable of quantitative analysis of hundreds of targeted lipids at high sensitivity
in a single on-line chromatographic separation. Imaging mass spectrometry of lipids in tissues has opened new insights into
the distribution of lipid molecular species with promising application to study pathophysiological events and diseases.

Full text

New Applications of Mass
Spectrometry in Lipid Analysis
*
Published, JBC Papers in Press, June 1, 2011, DOI 10.1074/jbc.R111.233478
Robert C. Murphy
‡1
and Simon J. Gaskell
§
From the
‡
Department of Pharmacology, University of Colorado Denver,
Aurora, Colorado 80045 and the
§
Queen Mary University of London, Mile
End Road, London E1 4NS, United Kingdom
Mass spectrometry has emerged as a powerful tool for the
analysis of all lipids. Lipidomic analysis of biological systems
using various approaches is now possible with a quantitative
measurement of hundreds of lipid molecular species. Although
availability of reference and internal standards lags behind the
field, approaches using stable isotope-labeled derivative tagging
permit precise determination of specific phospholipids in an
experimental series. The use of reactivity of ozone has enabled
assessment of double bond positions in fatty acyl groups even
when species remain in complex lipid mixtures. Rapid scanning
tandem mass spectrometers are capable of quantitative analysis
of hundreds of targeted lipids at high sensitivity in a single on-
line chromatographic separation. Imaging mass spectrometry of
lipids in tissues has opened new insights into the distribution of
lipid molecular species with promising application to study
pathophysiological events and diseases.
Mass spectrometry has been applied to the analysis of lipids
since its early origins as an analytical tool for organic chemistry.
Fundamental studies of fatty acid esters proved that MS not only
could reveal detailed structural information from known com-
pounds (1) but also was highly useful for the structural elucidation
of unknown lipids such as the prostaglandins (2). Lipids also were
used as molecules to probe the basic mechanisms of ion fragmen-
tation following electron ionization (3). However, very few lipids
were amenable for direct MS analysis because of the absolute
requirement that the molecule must have a sufficient vapor pres-
sure to enter as a gas into the ion source of the mass spectrometer.
This remarkably changed when fast atom bombardment ioniza-
tion was first introduced (4), and nonvolatile lipids such as phos-
pholipids (5) could be directly analyzed; yet it was the develop-
ment of electrospray ionization (ESI)
2
by Fenn et al. (6) and
MALDI by Karas and Hillenkamp (7) that truly opened the vast
array of lipids found in biology (Table 1) to direct analysis by
MS. The situation today is that all lipids are amenable to MS
and the numerous ancillary techniques engaged in current
practice.
These desorption and spray ionization techniques surpris-
ingly impart very little energy to the ionized lipid, and thus,
protonated molecular ions ([M ⫹H]
⫹
) or adducted molecular
ions (such as [M ⫹NH
4
]
⫹
,[M⫹Na]
⫹
, and [M ⫹K]
⫹
) are the
usually observed cations. Deprotonated molecular anions
([M ⫺H]
⫺
) and adducted anions ([M ⫹Cl]
⫺
and [M ⫹ace-
tate]
⫺
) are observed if the molecule preferentially forms nega-
tive ions. At the same time that these remarkable ionization
techniques were being developed, tools were emerging to
impart energy to break covalent bonds within ions using tech-
niques such as collisional activation and collision-induced dis-
sociation (CID) and to transmit product ions using efficient
collision cells (radio frequency-only quadrupole fields) and
then reanalyze the product ions by a second mass spectrometer
(MS/MS). The triple quadrupole mass spectrometer encom-
passed these advances of CID and was found to be well suited
for the analysis of lipids through its many modes of MS/MS
operation, including product ion scanning, precursor ion scan-
ning, neutral loss scanning, and selected reaction monitoring
(SRM; also termed multiple reaction monitoring). High resolu-
tion mass analysis of molecular ion species and product ions
after CID became routinely possible with the second generation
time-of-flight analyzers (8) and ion-trapping technology of the
ion cyclotron resonance cell and the orbitrap mass spectrome-
ter (9). Much of this instrumental development occurred in the
commercial manufacturing sector because of the large market
for these tools in proteomic research. Nevertheless, these tools
were well suited for lipid analysis, and application of MS and
MS/MS to solve challenging problems was now possible.
Lipids are somewhat different from biomolecules such as
peptides, oligonucleic acids, and oligosaccharides from many
standpoints of MS analysis. Lipids are hydrophobic as well as
hydrophilic molecules (10), and the hydrophobicity typically
means a large number of –CH
2
groups or a large number of
hydrogens in the molecule that impart a significant mass defect
observed as the fraction of exact mass following the integer
mass of the molecular ion species. Information related to chem-
ical structure can often be obtained by CID, but this informa-
tion is encoded in the gas-phase ion chemistry of the lipid. As a
result of these developments in MS, which were largely realized
a decade or more ago, recent applications of MS to lipid analysis
have resulted in remarkable advances in our understanding of
lipid biochemistry.
One advance for lipid MS, perhaps not fully appreciated, has
been the increased availability of sophisticated mass spectrom-
eters in biochemical laboratories to study lipid biochemistry. A
specific example was the use of MS to refine our understanding
of the biological role of the gene product SRD5A3, which was
previously annotated as steroid 5
␣
-reductase type 3, and the
involvement of this protein in a severe congenital glycosylation
genetic disorder affecting humans (11). These investigators
were able to identify increased levels of polyprenol lipids rela-
tive to dolichols in the plasma of affected humans that lacked
*This work was supported, in whole or in part, by National Institutes of Health
Grant HL25785 and LIPID MAPS Consortium Grant GM06338. This is the
second article in the Thematic Minireview Series on Biological Applications
of Mass Spectrometry. This minireview will be reprinted in the 2011 Mini-
review Compendium, which will be available in January, 2012.
1
To whom correspondence should be addressed. E-mail: robert.murphy@
ucdenver.edu.
2
The abbreviations used are: ESI, electrospray ionization; CID, collision-in-
duced dissociation; SRM, selected reaction monitoring; TAG, triacylglyc-
erol; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidyl-
glycerol; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC,
glycerophosphatidylcholine; LAT, lysophospholipid acyltransferase; IMS,
imaging MS; SIMS, secondary ion MS; IM-MS, ion mobility-MS.
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this gene relative to normal subjects and were able to go on and
define SRD5A3 as an NADPH-dependent reductase that satu-
rates the
␣
-isoprene unit of polyprenols to yield the dolichol
structure. Because dolichols are required for N-linked glycosy-
lation in the Golgi apparatus, the loss of this rate-limiting
enzyme reduced overall glycosylation of proteins critical for
proper function. A MS method for the analysis of dolichols was
a recent result of having advanced instruments available for
lipidomic studies in laboratories of lipid biochemists (12).
Lipidomic Studies
One of the significant changes that have occurred has been
an understanding of lipids as they exist as complex mixtures
within the cellular environment. This has naturally led to the
concept that revealing the nature of this mixture is an impor-
tant goal in understanding cell biology or system biology. This
has resulted in the widespread use of the term “lipidomics,”
which unfortunately means different things to different indi-
viduals. On one hand, some have a “global” view of lipidomics as
the identification and quantitation of all lipids in a cell (tissue or
organism) and studies of how they vary with state/challenge of
the cell. Others use the term to define quantitative analysis of a
restricted group such as the major lipids present in a biological
sample. The sheer number of actual lipid substances within
living cells is enormous (13). The identified and quantitated
lipids within the macrophage have been reported to be as high
as 1000 individual molecular species with caveats that more
species were detected than could be quantitated (14). The
dynamic range in which lipid concentrations can vary in a tissue
may be 10
6
or more (from nanomolar fatty acids to attomolar
eicosanoid lipid mediators), and this range challenges the capa-
bility of any single approach of analysis. As noted above, molec-
ular weight alone is insufficient to absolutely define a lipid
structure because this single measure introduces assumptions
as to identification. Identification by chromatographic reten-
tion time, measurement at high resolution (elemental compo-
sition at submillimass unit accuracy), gas-phase chemical
behavior, and elution with an isotope-labeled internal standard
may still be insufficient to absolutely identify a complex lipid
because of ambiguities arising from regio- or stereoisomerism
(sn-substitution for phospholipids or double bond position and
geometry for any fatty acyl substituent). Perhaps most insidious
is that the criteria employed to identify a lipid are often not
stated, which can also lead to misunderstandings on the part of
the reader. Nevertheless, practicality demands that assump-
tions have to be made because even state-of-the-art MS is insuf-
ficiently powerful to deal with the complex mixture of lipids in
a lipidomics study, yet one must balance the need to achieve
results.
One aspect of cellular lipid mixture complexity is that fami-
lies of closely related lipids are present that differ by the number
of fatty acyl carbons and number of double bonds (as well as
position of double bonds) as well as minor variants in structure
such as ether substitution for esters. These closely related fam-
ilies comprise molecular species that have very similar struc-
ture but often different molecular weights. Molecular species
that differ by common chain elongation and the number of
double bonds in the fatty acyl chain appear at intervals 24–28
Da higher or lower in the mass spectrum of the mixture. Many
of the lipids presented in Table 1 exist within cells as mixtures
of individual molecular species, and the MS experiment allows
one to readily discover those molecular ions species present in
an extract by either positive and/or negative ion MS. There
have been several reviews on the behavior of each of the lipid
classes and the individual molecular species in terms of their
CID ion chemistry after ionization by either electrospray or
MALDI (15–18). The challenge has been to analyze the nature
of the complex mixture and understand whether information
collected about different molecular species reveals unique bio-
chemical events or possibly pathophysiology.
There have been several approaches to deal with complex
mixtures of molecular species. The approaches taken can be
divided into two general methodologies. The first involves little
pre-separation of the lipids other than by simple solvent extrac-
tion, followed by direct analysis as the complex mixture of
many different lipid classes. This approach is called the “shot-
gun” method and certainly has specific advantages as well as
some disadvantages. The second approach has been to use
chromatographic separation of crude lipid extracts to isolate
specific lipid classes prior to analysis (Table 1) with or without
additional separation of the individual molecular species. For
example, normal-phase chromatography (which is a separation
method based on analyte molecular polarity) is well suited to
separate each of the lipid classes such as glycerophospholipids
from glycerolipids (triacylglycerols (TAGs) or diacylglycerols).
However, molecular species are not typically separated by this
technique, and to achieve this, reversed-phase HPLC is
employed. If individual classes of lipids are not pre-separated
through a normal phase-type approach, then separation only by
reversed-phase chromatography separates by lipophilicity and
is largely not influenced by the polar headgroup and the overall
polar character of the lipid.
The shotgun technique has been significantly refined to cap-
italize on the anionic and weakly anionic nature of many lipid
classes to optimize the analysis of highly different lipid classes
present in an extract (19). Direct analysis of the negative ions
formed by electrospray can reveal the presence of the very
acidic lipids such as phosphatidylinositol (PI), phosphatidylser-
ine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA),
cardiolipin, and sulfatides (sphingolipids) by the addition of a
base such as LiOH. Negative ion ESI can also yield signals from
the phosphatidylethanolamine (PE) molecular species. Positive
ion ESI-MS yields abundant [M ⫹H]
⫹
(or [M ⫹Li]
⫹
if LiOH is
added) for glycerophosphatidylcholine (PC) and glycerolipid
TABLE 1
Lipid categories and examples of molecular species
A complete list of lipids and classification numbers can be found at the Nature/
LIPID MAPS website. SM, sphingomyelin.
Category Specific lipid species
Fatty acyls Fatty acids, eicosanoids, endocannabinoids
Glycerolipids TAGs, diacylglycerols
Glycerophospholipids PC, PE, PI, PS, PG, PA, cardiolipin
Sphingolipids SM, sulfatides, sphingosine, ceramides, ganglioside
Sterol lipids Cholesterol, estradiol, testosterone, bile acids
Prenol lipids Farnesol, dolichols, vitamin K
Saccharolipids Lipid A, acyltrehaloses
Polyketides Aflatoxins, tetracyclines, erythromycin
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molecular species present in a complex mixture. Among the
potential confounding features of shotgun lipidomics is ion
suppression during the electrospray process that limits the abil-
ity to detect minor lipid species that may be present (20) and the
fact that any observed m/zmay contain multiple molecular ion
species. The shotgun method has been used for the quantita-
tion of specific lipids using techniques such as neutral loss and
precursor ion scanning (MS/MS) and internal standard/refer-
ence standard calibration protocols to generate standard curves
to convert ratios of target lipid ion abundance/internal stan-
dard ion abundance to absolute concentration of lipids in the
electrospray solvent system (21). The major advantages of the
shotgun method are the ease of implementation and the broad
coverage of major lipid molecular species that can be quanti-
tated in a relatively short period of time by a single laboratory.
Pre-separation of a lipid extract using specific chromato-
graphic protocols developed for each lipid class can enable the
identification of far more lipid species that differ in subtle ways.
The analysis of the neutral lipids from RAW 264.7 cells by nor-
mal-phase chromatography followed by LC-MS enabled the
detection of ether-linked triradylglycerols because they were
separated by polarity from the TAGs (22). An ether-linked tri-
radylglycerol such as 16:0 ether/18:0/18:1 differs only slightly
(0.027 thomson units) in the mass/charge ratio of the observed
[M ⫹NH
4
]
⫹
from an odd-chained analog such as 15:0/18:0/
18:1 TAG but substantially differs in mobility under the nor-
mal-phase chromatographic separation employed. These
ether-linked glycerides could have easily been misidentified
without a separation step. As with shotgun lipidomics, this
LC-MS and LC-MS/MS approach for lipid analysis can be set
up for quantitative analysis using internal standards and cali-
bration curves established with reference standards (22). The
chromatographic pre-separation approach was used to identify
⬎1000 different molecular species recently measured in the
macrophage lipidome (14). To achieve this quantitative meas-
ure for this large number of molecular species, it was necessary
to separate and to employ a different separation protocol that
was ideal for each individual class of lipid. Lipidomic analyses of
yeast (23), Caenorhabditis elegans (24), tuberculosis mycobac-
teria (25), human plasma (26), and others (27) have emerged.
Challenges still remain in the lipidomics area, in large part
because it is very tedious to establish and carry out the quanti-
tative analysis method. However, more importantly, there are
insufficient internal as well as few reference standards commer-
cially available that can be used to generate the appropriate
calibration curves to convert abundance of ions into a quanti-
tative measure of lipid concentration for many lipid classes. The
abundance of any molecular ion species, e.g. [M ⫹H]
⫹
, typi-
cally carries information on concentration, but ion abundance
is confounded by a number of features, including instrument
response factors, ionization efficiency of the molecule, stability
of the molecular ion species, and the presence of other mole-
cules that could cause ion suppression of the analyte of interest.
Quantitative MS has evolved to the highly specific and sensitive
“gold standard” level of acceptance because of measures to
avoid many of these problems. The central strategy of isotope
dilution has been to use a stable isotope-labeled analog of the
analyte as an internal standard and to measure the ratio of sig-
nal intensities of the analyte and internal standard rather than
any absolute intensity. The ratio is then converted to analyte
concentration by a calibration curve generated using reference
standards. However, this approach is not available for most lip-
idomic studies because of the absence of internal standards for
each and every lipid molecular species and, practically, the
potential overlap of m/zbetween internal standards and endog-
enous lipids. The absence of reference standards for the differ-
ent variants of structure observed in a molecular species series,
e.g. number and position of double bonds in fatty acyl chains,
variety of fatty acyl chain length, ether analogs, even positional
isomers for phospholipids and glycerolipids, further compli-
cates analysis because one cannot easily measure parameters
such as ionization efficiency, ion stability, and response factors
that are critical for the accuracy of the quantitative analysis.
Often, quantitation is based on an “average standard curve” or,
worse, just a single reference standard for many different struc-
tural variants in a series. Nevertheless, given the problems with
accuracy, these measurements remain precise and useful when
comparing changes of the same lipid molecular species within
an experimental series.
An alternative approach to absolute quantitation has been to
express the abundance of molecular species within a single class
of lipids as mole fraction using the abundance ratios of all
molecular species in that class to normalize the data. Relevant
information can be readily gleaned as exemplified in an exten-
sive report of phospholipid molecular specie measurements in a
study of lysophosphatidic acid acyltransferases that supply
polyunsaturated fatty acids to phospholipids that become
incorporated into cellular membranes (28).
Stable Isotope Tagging
An alternative approach for aminophospholipid quantita-
tion within a complex mixture of molecular species had its ori-
gins in the growing need for quantitation in peptide analysis. In
this case, phospholipids such as PE and PS are derivatized with
stable isotope-labeled reagents that tag the molecule within a
specific treatment series. For example, identical aliquots of cells
are treated under conditions A, B, C, and D. Each cell treatment
aliquot is derivatized with a different stable isotope internal
standard to tag each incubation condition. The tag is then used
to decode the lipid molecular species as to each experimental
condition, and the ratios of isotopically distinct ion signal
intensities allow determination of changes in concentration.
This multiplexed quantitative approach was first demonstrated
with commercial derivatization iTRAQ (isobaric tag for relative
and absolute quantitation) reagents, in which product ions of
derivatized aminophospholipids were analyzed but required
MS
3
to decode (29). An alternative derivative was developed
using four different stable isotope-labeled dimethylaminoben-
zoic acid N-hydroxysuccinimide esters and precursor ion scan-
ning to decode modified or newly formed aminophospholipids
in cells treated with ozone (30). These studies readily revealed
the differences in concentrations of PE molecular species
because the ionization phenomenon was virtually identical for
all of the products under analysis, and in effect, a stable isotope
variant was made for each aminophospholipid molecular
species.
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Fatty Acyl Double Bond Positional Analysis
The exact position of a double bond in a fatty acyl chain
remains rather difficult to determine by MS alone, yet impor-
tant information can be ignored if such determinations are not
made. For example, vaccenic acid (18:1(n-7)) is a common
trans-fatty acid in cow’s milk, but it is isomeric in terms of
double bond position to oleic acid (18:1(n-9)), the more com-
mon monounsaturated fatty acyl group found in phospholipids,
sphingolipids, and glycerolipids. Thus, the identification of an
18:1 fatty acyl group, for example, by neutral loss scanning or
product ion scanning in lipidomic analyses is ambiguous with
respect to positional isomers. Recently, a method using ozone
to effect carbon bond cleavage in phospholipids was introduced
and revealed that often this fatty acyl group is a mixture of n-7
and n-9 fatty acids (31, 32). This ozone approach has been
expanded to include collisional activation of ions in the pres-
ence of ozone (combined CID-ozone identification), which can
lead to product aldehyde ions that reveal the double bond posi-
tion (Fig. 1).
Targeted Lipid Quantitation
The quantitation of eicosanoids and other lipid mediators by
MS was well established even when only GC-MS and negative
ion chemical ionization were available (33). The analysis of
these lipids has always been a challenge because of the low
quantities made within tissues or cells. LC-MS/MS has
emerged as the technique of choice to study these molecules
because no derivatization is required, and this class of arachi-
donic acid metabolite has a free carboxylic acid moiety that
renders efficient negative ion formation (carboxylate anion) by
ESI. The recent advances in MS instrumentation include rapid
scanning; for example, in the single reaction monitoring mode
of MS/MS operation, it is now quite possible to monitor 20 –50
selected ion transitions with a duty cycle of less than a second.
The recent advances in chromatography at high pressure
(ultra-HPLC) have greatly reduced retention times, and the
combination of ultra-HPLC with rapid SRM has enabled devel-
opment of methods to target not one eicosanoid in a single
LC-MS/MS assay but 100 eicosanoids at high sensitivity and
specificity (34).
Rapid SRM scanning was also used in the development of an
enzyme choice assay to characterize newly discovered lyso-
phospholipid acyltransferases (LATs) (35). Enzymatic proper-
ties such as substrate specificity have hitherto often been deter-
mined using radiolabeled tracers and Michaelis-Menten
kinetic parameters. An alternative approach was recently taken
that relied on the ability to rapidly determine the reaction prod-
ucts when mixtures of potential substrates were mixed with
cloned enzymatic proteins. In this particular case, four different
LATs were studied by the addition of a mixture of six lysophos-
pholipid subclasses (choline, ethanolamine, inositol, glycerol,
serine, and lyso-PA) with eight different CoA esters. This
resulted in 48 potential products, requiring (after inclusion of
controls) 60 SRM ion transitions to monitor during an LC-
MS/MS run. Remarkable substrate specificity was observed in
this competition assay for each of the different LATs of the
MBOAT (membrane-bound O-acyltransferases) family (35) as
well as three unique LATs from Drosophila (Fig. 2) (36). A sim-
ilar mixed substrate approach to assess enzymatic activity was
also used in studies of LPAAT3 (lysophosphatidic acid acyl-
transferase 3) (28).
Imaging MS (IMS) of Lipids
An area of very rapid growth has been the imaging of tissues
by MS. Studies of lipid biochemistry have always suffered from
the inability to determine the location of specific lipid sub-
stances at or near the cellular level. IMS partially bridges this
gap but has the additional advantage of MS specificity. Several
different ionization methods have been explored to generate
images of the distribution of lipid species as they occur in tis-
sues. Secondary ion MS (SIMS) has historically been the
method used to generate lipid-derived ions, but the energy of
this technique often results in lipid structural damage and loss
of molecular species information (37). Nonetheless, this tech-
nique has the best lateral resolution (⬍50 nm is possible) and is
well adapted to specific lipid experiments such as tracing the
transport of
13
C-labeled oleate in an adipocyte (Fig. 3, Aand B),
where the ion observed is
13
C
⫺
(38). Recent advances using
caged C
60
⫹
(buckyballs) as projectile ions have enabled intact
lipids (cholesterol) to be imaged in the SIMS experiments and
suggest that it may be possible to image abundant lipids at cel-
lular resolution (Fig. 3, C–E) (39).
MALDI can be readily coupled with imaging of lipids in tis-
sues because the most abundant ions released in this MALDI-
IMS experiment are the lipids that make up the cellular mem-
branes and are in lipid droplets. Remarkable images are
appearing as to the regional distribution of lipids in tissues such
FIGURE 1. Ozone reaction with phospholipids in the collision cell of a
linear ion trap mass spectrometer. a, ESI mass spectrum obtained by direct
infusion of a crude lipid extract from cow brain. b, combined CID-ozone iden-
tification mass spectrum acquired by applying collision energy to the mass-
selected precursor ion at m/z 782.6 with ozone vapor present in the collision
cell (q2). c, molecular structure of four regioisomeric lipids that could give rise
to the combined spectral features observed in b. This figure has been
reprinted with permission from the American Chemical Society (31).
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as brain (40) and kidney (41) as well as large biological struc-
tures such as embryos (42) and entire organisms such a mouse
(43). The secondary lipid ions that are observed do correlate
fairly well with local concentrations of specific lipids that are
readily desorbed and ionized by this technique. This correlation
is particularly high for PC in the positive ion mode (44) and for
PI and sulfatides in the negative ion mode. However, there is
bias in this experiment in that some lipids known to be present
are not easily observed, perhaps due to ion suppression. For
example, PE molecular species are not readily observed in the
positive ion mode in some instruments, possibly due to their
rather facile loss of the polar headgroup (141 Da) to yield posi-
tively charged diglyceride-like ions. Another curious feature is
that the phospholipids and glycerolipids are observed as alkali
metal adducts (Na
⫹
and K
⫹
) as well as the protonated species.
Recent reports (51) have suggested that there is information in
the relative abundance of these ions that reflects edema and loss
of the ATPases that maintain the cellular cation gradient (Fig. 3,
FIGURE 2. Dual choice LAT enzyme assay by selected ion recording and LC-MS/MS. The fly MBOAT proteins Oys, Frj, and Nes were expressed in the
acyltransferase-deficient ale1⌬yeast strain. Isolated microsomes containing the fly proteins were incubated with a mixture of eight acyl-CoA species and six
lysophospholipids, and the products formed by each enzyme were separated and quantified by LC-MS/MS. Scales were adjusted to highlight the substrate
preferences of each enzyme. Results given are the mean ⫾S.E. of three experiments. Acyl-CoAs are abbreviated as x:y, where xis the number of carbon atoms
in the chain and yis the number of double bonds. This figure has been reprinted with permission from the American Society for Cell Biology (36).
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Fand G). Another ionization technique, desorption ESI, has
also been successfully used to image lipids in various tissues,
including brain (45, 46).
Combined Ion Mobility-MS (IM-MS) of Lipids
The coupling of ion mobility separation with either single
stage or tandem MS provides an additional dimension of anal-
ysis on a far more rapid time scale than achieved with conven-
tional pre-separation of lipid classes using condensed-phase
chromatographic methods. Woods and co-workers (47)
reported a systematic study of several polar lipid classes, illus-
trating distinct behaviors in the relationship between ion
mobility and m/zratio. The combination of MALDI and
IM-MS was exploited by Kliman et al. (48) in examining intact
brain tissue from Drosophila; quantitative comparisons of dif-
ferent tissue samples were followed by IM-MS/MS analyses to
allow identification of those molecular species present in differ-
ing amounts. Further studies by Trimpin et al. (49), this time
using ESI, provided evidence for phospholipid aggregates in the
gas phase, interpreted as inverted micelles, which were sepa-
rated by IM-MS prior to disaggregation and further mobility
analysis. Three stages of IM-MS interspersed with activated
decomposition also allowed distinction between sn-1 and sn-2
isomers, illustrated using PGs. Such sophisticated analyses,
employing tandem ion mobility coupled with MS, are unlikely
to become routine, but the improved separation power of com-
bining single stage mobility separation and MS or MS/MS is
such that widespread application is likely.
Conclusion
Over the past decade, significant advances have been made in
MS instrumentation that significantly impact the analysis of
lipids in biological samples. Sensitivity for the LC-MS detection
of lipids has increased perhaps 50–100-fold with the develop-
ment of advanced tandem quadrupole mass spectrometers as
well as advanced LC technology. Along with this increased in
sensitivity has come increased scanning speed, which permits
almost unlimited selected ion recording of ion transitions when
coupled with computer-driven timing of the SRM experiments.
Remarkable advances in IMS of lipids will likely drive the devel-
opment of improvements in rastering laser beams for the
MALDI mass spectral data acquisition stage. The expected
application of ion mobility technology to real problem solving
in lipid biochemistry will likely prove the value of this MS tech-
FIGURE 3. Imaging of lipids using MS. A, SIMS imaging of fatty acid transport in cultured adipocytes after unwashed 3T3F442A adipocytes were incubated
with [
13
C]oleate. Images are of
13
C
⫺
.Scale bar ⫽5
␮
m. This figure has been reprinted with permission as open access from Ref. 38. B, optical image using a
reflection differential interference contrast microscope of the same cells before analysis with SIMS. Reflection differential interference contrast (DIC) images
(magnification ⫻500) were obtained using a Nikon Eclipse E800 upright microscope. Scale bar ⫽5
␮
m. This figure has been reprinted with permission as open
access from Ref. 38. C, scanning ion image of an axial slice from a 9-day-old mouse embryo. The gut and genital ridge (GR) are identified by white arrows.Dand
E, SIMS images of cholesterol (m/z 366 –370) from the tissue in the SIMS image. The image in Ewas taken before a sputter dose of 1 ⫻10
13
C
60
⫹
60
⫹
/cm
2
, and the
image in Cwas taken after nanotome sputtering. This figure has been reprinted with permission from Elsevier (39). Fand G, rat brain sections from a traumatic
brain injury model and imaging by MALDI-IMS corresponding to 16:0/18:1 PC ([M ⫹Na]
⫹
,m/z782.6) and 16:0/18:1 PC ([M ⫹K]
⫹
,m/z798.6), respectively (51).
MINIREVIEW: New Applications of Mass Spectrometry
25432 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 •NUMBER 29 •JULY 22, 2011
at Univ Colorado - Denison Memorial Library on January 14, 2015http://www.jbc.org/Downloaded from

nology, which is still in its infancy. The utility of ion-trapping
instruments and, in particular, the newer technologies of high
energy collision in the orbitrap promise the re-emergence of
capability to carry out charge-remote decomposition experi-
ments useful for positional analysis of double bonds in phos-
pholipids and glycerolipids (50). In the midst of these techno-
logical advances are the advances in lipid biochemistry that can
result.
Acknowledgments—R. C. M. thanks the Queen Mary University of
London and the William Harvey Institute for access to resources used
in the preparation of this minireview.
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Lipid Analysis
New Applications of Mass Spectrometry in
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