annurev-biochem-060409-092612

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Applications of MassSpectrometry to Lipidsand Membranes

Richard Harkewicz and Edward A. Dennis

Department of Chemistry and Biochemistry and Department of Pharmacology,School of Medicine, University of California at San Diego, La Jolla, California 9209email: [email protected], [email protected]

Annu. Rev. Biochem. 2011. 80:30125

First published online as a Review in Advance onApril 5, 2011

The Annual Review of Biochemistry is online at

biochem.annualreviews.org

This articles doi:10.1146/annurev-biochem-060409-092612

Copyright c 2011 by Annual Reviews.All rights reserved

0066-4154/11/0707-0301$20.00

Keywords

CLASS, DXMS, imaging mass spectrometry, lipidomics, novel lip

shotgun

AbstractLipidomics, a major part of metabolomics, constitutes the detailedysis and global characterization, both spatial and temporal, of the s

ture and function of lipids (the lipidome) within a living system

with proteomics, mass spectrometry has earned a central analyticain lipidomics, and this role will continue to grow with techno

cal developments. Currently, there exist two mass spectrometryblipidomics approaches, one based on a division of lipids into categ

and classes prior to analysis, the comprehensive lipidomics anaby separation simplification (CLASS), and the other in which all

species are analyzed together without prior separation, shotgun. I

ploring the lipidome of various living systems, novel lipids are bdiscovered, and mass spectrometry is helping characterize their chcal structure. Deuterium exchange mass spectrometry (DXMS) is b

usedtoinvestigatetheassociationoflipidsandmembraneswithpro

and enzymes, and imaging mass spectrometry (IMS) is being applithe in situ analysis of lipids in tissues.

301

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 302MASS SPECTROMETRYBASED

LIPIDOMICS . . . . . . . . . . . . . . . . . . . . . 303Lipid Definition and Classification. . 303

Sample Preparation for Mass

SpectrometryBased LipidomicsStudies . . . . . . . . . . . . . . . . . . . . . . . . . 303

Mass Spectrometers Optimal for

Lipid Analysis . . . . . . . . . . . . . . . . . . 307Mass Spectrometric Analysis: The

Shotgun Approach . . . . . . . . . . . . . . 308Comprehensive Lipidomics Analysis

by Separation Simplification . . . . . 309

MASS SPECTROMETRYBASEDSTRUCTURE

DETERMINATION OF

N O V EL LIPID S . . . . . . . . . . . . . . . . . . 3 1 1Targeted versus Untargeted

Lipidomics . . . . . . . . . . . . . . . . . . . . . 311

Examples of Novel, UnexpectedLipids . . . . . . . . . . . . . . . . . . . . . . . . . . 312

Untargeted-Flagged Lipidomics . . . . 312PROTEIN/ENZYME

ASSOCIATION WITH LIPIDS

AND MEMBRANES. . . . . . . . . . . . . . 315Peptide Amide Hydrogen/

Deuterium Exchange Mass

S pectr om etr y . . . . . . . . . . . . . . . . . . . 3 1 5Association of Proteins/Enzymes

with Lipids and Membranes . . . . . 316

CELL/TISSUE IMAGING OFLIPIDS BY MASS

SPECTROMETRY . . . . . . . . . . . . . . . 317Localization of Lipids in Tissues

and Cells . . . . . . . . . . . . . . . . . . . . . . . 317Matrix-Assisted Laser Desorption

Ionization-Imaging Mass

S pectr om etr y . . . . . . . . . . . . . . . . . . . 3 1 7

Future Directions in ImagingMass Spectrometry. . . . . . . . . . . . . . 318

SUMMARY AND OUTLOOK . . . . . . . 320

INTRODUCTION

The exponential growth in the number of fu

sequenced genomes available in the public d

main has provided biologists with the challenof connecting genes to gene function, that

genotype to phenotype, and to determine tactual role of genes. The drive for understan

ing the function of newly discovered genes hled biologists toward the systematic analysis

expression levels of the components that costitute a biological system, i.e., the mRNA, t

proteins, and the metabolites, and the globcataloging of these components hasgiven rise

the various OMEs (the genome and the prteome). Additionally, understanding networ

and how these different components intera

with one anotherbe it within a specific OMor between themis the basis of a systems b

ology approach (1, 2).Metabolomics is the systematic study of t

complete set of the nonproteinaceous, lomolecular-weight, endogenously synthesiz

intermediates (the metabolome) contained the cell (3, 4) and, it can be argued, repr

sents the end product of gene expression. Tmetabolome most closely correlates to an o

ganisms actual phenotype and hence has dease implications. Of all the molecules co

tained in the metabolome, the fats (or lipid

constitute the largest subset, including tens thousands of distinct lipid molecular speci

existing in the cells and tissues. Lipidomicsdominant part of metabolomics, is the detail

analysis and global characterization, both sptial and temporal, of the structure and fun

tion of lipids (the lipidome) within a living sytem. Comparing the lipidome of healthy vers

diseased states can provide information helful in correlating the role of lipids in vario

diseases, such as cancer, atherosclerosis, an

chronic inflammation. Additionally, a large vriety of lipid species comprise cellular mem

branes, and investigating their interactions wimembrane-associating proteins/enzymes c

provide insight into such areas as drug/inhibitinteractions.

302 Harkewicz Dennis

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The very first mass spectrometer was built

by Nobel Laureate Sir J.J. Thomson (5) in theearly part of the twentieth century and was used

to analyze marsh gas. In his analysis, Thom-son observed the mass-to-charge ratios (m/z)

of 16 and 26, which he identified as the posi-

tiveions of methane andacetylene, respectively.

Since its very first inception, mass spectrometryhas played a central analytical role in the var-ious sciences, and its contribution during the

past few decades to the fields of proteomics andmetabolomics cannot be overstated.

A number of excellent reviews featuringmass spectrometry as well as its applications

in the various OMIC disciplines already ex-ist (610), and the reader is encouraged to ex-

plore these resources. Griffiths & Wang (11)recently wrote a particularly informative review

that covers mass spectrometry applications in

proteomics, metabolomics, and lipidomics.Our present review presents a critical

discussion of the current status of applicationsof mass spectrometry to the field of lipidomics,

and we do not wish to simply repeat what isalready available. Thus, four main areas are

covered. First, we compare and contrast twocurrent mass spectrometrybased lipidomics

approaches, one where the lipidome is dividedinto lipid categories and classes and each

analyzed separately and the other where alllipid species are analyzed essentially together.

Second, we review the mass spectrometry

based structure determination of novel lipids.Third, the current state of deuterium exchange

mass spectrometry (DXMS) used to study thelocation and orientation of proteins associated

with lipid membranes is covered. Lastly, wereview advances in the imaging of lipids in tis-

sues, including matrix-assisted laser desorptionionization (MALDI) mass spectrometry.

MASS SPECTROMETRYBASEDLIPIDOMICS

Lipid Definition and Classification

Examination of a typical biochemistry dictio-

nary (12), university biochemistry textbook

(13), or volume on lipid analysis (14) for a

description of lipids, yields a broadly definedgroup of organic compounds, including the

fats, oils, waxes, sterols, and triglycerides, which are insoluble in water but soluble in

nonpolar organic solvents, are oily to the touch,

and together with carbohydrates and proteins

constitute the principal structural material ofliving cells. In reality, there are many examplesof lipids that do not adhere to this loose defini-

tion; this has recently led some in the scientificcommunity to define this most diverse set of

molecules on the basis of their biosyntheticorigin. The LIPID MAPS Consortium (15) has

defined lipids as hydrophobic or amphipathicsmall molecules that originate by carbanion-

based condensation of thioesters (fatty acids,polyketides, etc.) and/or by carbocation-based

condensation of isoprene units(prenols, sterols,

etc.). Adhering to this definition and in con-junction with the International Committee for

the Classification and Nomenclature of Lipids,the LIPID MAPS Consortium has thus defined

eight categories of lipids based on their chem-ically functional backbonefatty acyls, glyc-

erolipids, glycerophospholipids, sphingolipids,sterol lipids, prenol lipids, saccharolipids, and

polyketidesalong with numerous classesand subclasses to allow one to describe all

lipid molecular species (15, 16). Also, see theLIPID MAPSNature Lipidomics Gateway

at http://www.lipidmaps.org. Figure 1

illustrates examples of molecular species foreach of these eight lipid categories.

Sample Preparation for MassSpectrometryBasedLipidomics Studies

Two fundamentally different approaches ex-

ist for the mass spectrometrybased identifi-cation and quantification of lipids within cells

and tissues. The first approach, a more tradi-

tional comprehensive lipidomics analysis byseparation simplification (CLASS) strategy

and the platform used by the LIPID MAPSConsortium, is based on separation of dif-

ferent lipid categories using extraction and

www.annualreviews.org Mass Spectrometry of Lipids 303
http://www.lipidmaps.org/http://www.lipidmaps.org/http://www.lipidmaps.org/

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O

HO

O

HO

Fatty acyls (FA)[prostaglandin D2]9S,15S-dihydroxy-11-oxo-5Z,13E-prostadienoic acid

HO

HO

HO

HOHOHO

HO

OH

OH

H

H

H

H

H

H

OH

OH

OH

OH

OH

NH

OH

N+

H

O

O

O

O

O

O

O

OO

PO

O

O

O

O

O

O

P

O

OH

OH

NH

OH

OH

HO

HN

HO

O O

O

O O

O

OO

O

O

OON

O

O

O P P

O

Glycerolipids (GL) [DAG(16:0/18:1(9Z))]1-palmitoyl-2-oleoyl-sn-glycerol

Glycerophospholipids (GP) [PC(16:0/18:1(9Z))]1-palmitoyl-2-oleoyl-sn-glycerophosphocholine

Sphingolipids (SP) [Ins-1-P-Cer(t18:0/26:0)]N-(hexacosanoyl)-4R-hydroxysphinganine-1-phospho-(1 '-myo-inositol)

Prenol lipids (PR) [farnesol]2E,6E-farnesol

Sterol lipids (ST) [cholesterol]cholest-5-en-3-ol

Saccharolipids (SL) [UDP-2,3-diacyl-GlcN]UDP-2,3-(3-hydroxy-tetradecanoyl)-D-glucosamine

Polyketides (PK) [alfatoxinB1]cyclopenta[c]furo[3',2':4,5]furo

[2,3-h][1]benzopyran-1, 11-dione,

2,3,6a,9a-tetrahydro-4-m,(6aR-cis)

Figure 1

Examples of each of the eight categories of lipids as defined by the Lipid Metabolites and Pathways Strategy (LIPID MAPS)Consortium.


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Directinfusion

GC LC

Cells or tissues

Probe

Medium

Homogenate

Sonicate

Extract

Internal standards

Extraction

3. Additives (for ionization)

4. Mass spectrometer monitoring mode

CLASS Shotgun

Variables1. Mass spectrometer types

2. Ionization mode

Cellstissues

Figure 2

Flow chart depicting the lipid sample preparationand analysis protocol. After extraction, thecomprehensive lipidomics analysis by separationsimplification (CLASS) or shotgun lipidomicsapproach is carried out. GC, gas chromatography;LC, liquid chromatography.

chromatographic separation prior to mass anal-

ysis and then optimizing the mass spectrome-ter to perform in a lipid classspecific fashion.

The second approach, sometimes termed shot-gun lipidomics, omits chromatographic separa-

tion and essentially analyzes all the lipid classestogether, directly infusing them into the mass

spectrometer, while employing different ion

source polarities (to form positive or negativeions) and infusing ionization solution additives,

which serve to provide a kind of lipid classspecific favored analysis. Moredetailsregarding

each approach are provided below. For eitherapproach, a general cell or tissue preparation is

followed as outlined in Figure 2.Cells or tissues are cultured and can be sub-

jected to a probe (activation or perturbation),

as shown in Figure 2, and their lipidome is

compared to a control unperturbed sample. Forexample, a macrophage cell line can be sub-

jected to a bacterial endotoxin in a time/dose-response study (1719), or yeast cells can have

lipid synthesis genes altered or subjected to

different growth temperatures (20). Similarly,

human blood plasma (21) or tissue biopsies,for example, from ear tissue from normal ver-sus stressed states, can be analyzed (22). The

lipids stored within the cell wall andinternal or-ganelles or tissue matrix need to be released em-

ploying a disruption mechanism, such as sonica-tion, to produce a uniform homogenate. Some

lipids, such as eicosanoids (23), are actually notstored in the cell but secreted into the growth

medium, so the medium also needs to be saved

and analyzed for a complete lipidomics analy-sis (17, 19). Prior to extraction, it is necessary

to add a mixture of internal standards to en-able the absolute quantitation of lipids in the

sample. These internal standards are typicallydeuterium-labeled lipid analogs of the molecu-

lar species one wishes to quantitate. When suchdeuterium-labeled analogs are not available,

similar lipid class odd-carbon chain molecularspecies can be used. In either case, the goal is to

use an internal standard that the organism un-der study cannot possibly synthesize. Recently,

advances in the number and commercial avail-

ability of such lipid standards (24) have beena critical cornerstone to the growth and suc-

cesses of mass spectrometrybased lipidomics.Table 1 summarizes the lipid classspecific ex-

traction protocols optimized and used by theLIPID MAPS Consortium (2535). A recent

impressive shotgun lipidomics study, whichmeasured theyeast lipidome, actually employed

a two-step extraction procedure that separatedrelatively apolar and polar lipids (20). In all

these studies, the solvent containing extractswere then evaporated, and the remaining lipids

were resuspended in a liquid medium optimal

for direct infusion into the mass spectrome-ter (shotgun approach) or in a medium com-

patible with chromatographic separation priorto online mass spectrometric analysis (CLASS

approach). The types of mass spectrometers


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Table1

Extractionandchromatographicseparationproto

colsforlipidclasses

Lipidsp

eciesquantitatedc

Lipidcategory

Lipidclassa

Extractionb

Chroma

tographicseparationb

StudyAd

StudyBe

StudyCf

Fattyacyls

Fattyacids

MethanolicHCl/isooctane

Gaschro

matography

31

44

Eicosanoids

C18SPE

cartridge

HPLC/R

PC18column

76

9

Glycerolipids

DAG

Ethylac

etate/isooctane

HPLC/N

Psilicacolumn

55

107

TAG

18

105

Glycerophospholipids

PA

MethanolicHCl/chloroform

HPLC/N

Psilicacolumn

22

15

14

PC

26

36

26

PE

19

32

25

PS

24

20

26

PI

19

19

21

PG

22

16

21

Ether-linkedPC

5

7

9

Ether-linkedPE

10

13

9

N-acylPS

2

Cardiolipins

Methanol/chloroform

16

15

Sphingolipids

Ceramides

Incubationovernightinmethanol/

chloroformat48

Cfollowedby

methanolicKOH

HPLC/N

PNH2column

8

41

21

Dihydroceramides

8

12

Hexosylceramides

8

56

11

Dihydrohexosylceramides

8

9

Sphingomyelin

8

101

11

Dihydrosphingomyelin

8

9

Sphingoidbases

6

Sterollipids

Freeform

Methanol/chloroform

HPLC/R

PC18column

13

14

11

Fattyacylesters

Ethylac

etate/isooctane

HPLC/N

Psilicacolumn

22

23

Prenollipids

Ubiquinones

Methanol/chloroform

HPLC/R

PC8column

2

2

2

Dolichols

3

6

3

Totalnumberofspeciesquantitated

229

588

543

aDAG,diacylglycerols;TAG

,triacylglycerols;PA,glycerophosphate;PC

,glycerophosphocholine;PE,glycerophosph

oethanolamine;PS,glycerophosphoserine;PI,glycerophosphoinositol;PG,

glycerophosphoglycerol;HCl,hydrochloricacid;SPE,solidphaseextrac

tion;KOH,potassiumhydroxide;HPLC/RP

,reversed-phasehigh-performanceliquidch

romatography;HPLC/NP,

normal-phasehigh-perform

anceliquidchromatography.SeeReferences

15and16.

bSeeReferences2535forp

rotocoldetailsspecifictolipidclass.

cThethreecolumnsatrightshownumberofuniquelipidspeciesquantit

atedinthreeseparatestudies.

dSeeReference18.

eSeeReference21.


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being used for lipid analysis along with their

various modes of operations are mostly com-mon to both techniques and are described

next.

Mass Spectrometers Optimal forLipid Analysis

A mass spectrometric analysis of a moleculerequires that the molecule must be charged

either as a positive or a negative molecular ion.As such, the ion source is a fundamental com-

ponent of all mass spectrometers. The develop-ment of electrospray ionization (ESI) in the late

1980s (36), for which John Fenn was awardedpart of the 2002 Nobel Prize in Chemistry,

allows many molecules, including proteins,carbohydrates, and lipids, to be ionized in a

liquid medium without the need for prior de-

rivitization. ESI indeed revolutionized the fieldof mass spectrometry, allowing it to be used

for the analysis of molecules that could not beanalyzed in the past. ESI is used to form either

positive or negative ions, and some lipid classescan form either, both having their advantages

for determining molecular detail. It should benoted that ESI is a soft ionization technique,

and unlike electron impact ionization used formany years prior to the emergence of ESI, the

molecule is left whole and unfragmented inthe ionization process. ESI sources most com-

monly form positive molecular ions through

the addition of a proton to a molecule [M+H]+

or negative molecular ions through the re-

moval of a proton from a molecule [MH].Some large molecules, such as proteins, can be

multiply protonated (e.g., [M+H40]40+) usingESI. In such a case, a 40-kDa protein would

have an m/z of 1,000, well within the mass scanrange of most mass spectrometers. To increase

ionization efficiency for certain molecules, insome cases, a small amount of additive, such as

ammonium acetate, is added to the ESIsolution

to allow the formation of ammonium adductmolecular cations [M+NH4]+ or acetate

adduct molecular anions [M+CH3CO2].Because of its versatility, ESI is widely used for

all the lipid classes shown in Table 1 except

MS1 MS2Collision

cell

CID ScanningSelected m/z

Scanning CID Selected m/z

Selected m/z Selected m/z

Scanningm/z = x

CID

CID

Selectedm/z = x-a

Product-ion scan

Precursor-ion scan

Neutral-loss scan

Selected reaction monitoring

Figure 3

Various modes of tandem mass spectrometricanalyses available on the triple quadrupoleinstrument. Reprinted with permission fromReference 11. CID, collision induced dissociation.

for the free fatty acids; details regarding fatty

acid analysis are presented in a section below.The triple quadrupole mass spectrometer, a

very common workhorse in the mass spectrom-

etry field, is schematically diagramed in the toprow of Figure 3. The mass spectrometer canbe used in single-scan (MS) mode to provide

information about precursor molecular masses

within a specified mass range, but very oftenoperating the mass spectrometer in a tandem-

scan (MS/MS) mode can provide much moreuseful and additional information. It should be

noted that the triple quadrupole instrument is,but not all mass spectrometers are, capable of

operating in the MS/MS mode. In a typical

MS/MS experiment, a specified precursor ionis selected according to its mass-to-charge ratioin the first filter of the instrument and is frag-

mented into its product ions in a collision cell.

Then, the resulting product ions are scannedaccording to their mass-to-charge ratio in a sec-

ond filter, and a mass-to-charge ratio spectrumis recorded. This particular MS/MS scan mode


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is commonly termed product-ion scan and is

depicted in the second row ofFigure 3. Struc-tural isomers of some of the lipids, which pro-

vide the same precursor molecular mass in MSmode and are impossible to resolve, many times

yield different fragment product ions, allow-

ing them to be differentiated using the MS/MS

mode.The triple quadrupole instrument is capableof additional specialized MS/MS scan modes,

see Figure 3, and some of these are particu-larly useful in theanalysis of specific lipid classes

(2535). In the precursor-ion scan, the secondfilter is set to transmit only a specified ion frag-

ment, e.g., the [PH2O4C2H4N(CH3)3]+ phos-phocholine headgroup at m/z = 184, which is

characteristic of glycerophosphocholine lipids, while the first filter scans and produces a

mass-to-charge ratio spectrum for all precursor

molecular species producing this specific frag-ment. Another example of the precursor-ion

scan is when fatty acid cholesteryl esters are an-alyzed as ammonium adduct cations. The sec-

ondfilterissettotransmitonlythe[cholesterol-H2O]+-dehydratedcholesterolcationat m/z =369, the only fragment produced and a charac-teristic of fatty acid cholesteryl esters, while the

first filter produces a mass-to-charge ratio spec-trum of all precursor cholesteryl esters produc-

ing this fragment (37). In a neutral-loss scan,the first and second filters are scanned in uni-

son with a specified offset mass difference set

between them. As an example, a neutral-lossscan of 141 or 185 is used to monitor glyc-

erophosphoethanolamine or glycerophospho-serine lipids, respectively, accounting for the

neutral loss of phosphoethanolamine or phos-phoserine headgroups.

An ultrasensitive MS/MS mode on the triplequadrupole instrument is called selected reac-

tion monitoring (SRM). In SRM, the first filteris locked on a specified precursor ion, and the

second filter is locked on its specified product

ion. As described below, the SRM mode is par-ticularly useful when coupled with chromato-

graphic separations.Although the triple quadrupole mass spec-

trometer offers these specialized scan modes, it

is limited in its mass accuracy and resolutio

to 100 ppm and M/M 100, respectiveOther instruments used in lipidomics, such

the time-of-flight (TOF) mass spectrometand the Fourier transform orbitrap mass spe

trometer, can provide mass accuracies and re

olutions on the order of 520 ppm and 10

which in some cases is useful when determiing the elemental composition of lipid moleclar species, such as C42H81O9P (760.5618 D

versus C43H85O8P (760.5982 Da). Referenceprovides a helpful comparison of mass spe

trometer specifications (cost, size, mass accracy, mass resolution, etc.).

Mass Spectrometric Analysis:The Shotgun Approach

The shotgun lipidomics approach (20, 38)the name adapted from earlier shotgun g

nomics and shotgun proteomics approaches

hasproducedimpressiveresults to profilelipidincluding glycerophospholipids, glycerolipid

sphingolipids, and sterols, in biological extracby cleverly selecting the optimal ESI pola

ity, ESI solution additives, and tandem maspectrometer monitoring modes, as well as

developing and utilizing sophisticated analsis software to decipher the complicated da

output (39). Although the shotgun approais biased toward the ionization of molecul

species that are most abundant or those ha

ing the highest ionization efficiencies, whican cause ion suppression resulting in lo

level species not being detected, Han & Gro(38, 40) sought to minimize this drawba

by introducing the technique of intrasourseparation in which a crude biological e

tract is initially analyzed by ESI in the neative ionization mode, which favors ioniz

tion of anionic glycerophospholipids that anegatively charged at neutral pH (glyceropho

phoinositols, glycerophosphoglycerols, gly

erophosphoserines, glycerophosphates, cardolipins). Next, a base is added, such as LiOH

methanol, which favorsionizationof weakly aionic glycerophospholipids (glycerophosph

ethanolamines). Lastly, the extract is analyz


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in the positive ESI mode, which favors polar

lipids (glycerophosphocholines, sphingolipids).Intrasource separation greatly expands the dy-

namic range of lipidome observations, and us-ing this technique, the recorded mass spectra of

a crude extract from a mouse myocardium (40)

is shown in Figure 4a, where many classes of

glycerolipids, glycerophospholipids, and sphin-golipids were observed.In another example of shotgun lipidomics,

although yeast cells are only capable of synthe-sizing a limited number of fatty acids and other

more complex lipids compared to mammaliancells, Ejsing et al. (20) carried out an impressive

yeast lipidome study that quantified as manyas 162 molecular lipid species in a single yeast

strain and a total of 250 different species in allthe combined yeast strains studied (wild type

and mutants) covering 21 types of lipids; the

results of this study are shown in Figure 4b.

Comprehensive Lipidomics Analysisby Separation Simplification

The mammalian lipidome is estimated to

contain some hundreds of thousands ofmolecular species (2, 11), and compared to

the yeast lipidome, it is far more complex. To explore such an enormous lipidome, the

LIPID MAPS Consortium chose to developa comprehensive lipidomics analysis by sep-

aration simplification known as the CLASS

approach, which simplifies what is put into themass spectrometer by first chromatographi-

cally separating the components online andcoupling the chromatographic effluent directly

into the mass spectrometer. In short, thisconsitutes a divide-and-conquer strategy. An

advantage of CLASS is the minimization ofion suppression, which improves the ability to

detect very low-level lipid species, should theybe present. Table 1 summarizes the LIPID

MAPS CLASS approach and lists the lipid

classspecific chromatographic separation pro-tocols they have employed. Details for each of

these protocols are found in References 2535. All of the lipid classes, except the fatty

acids, in Table 1 were separated prior to mass

analysis, employing high-performance liquid

chromatography (HPLC). Historically, gaschromatography (GC) has been the method of

choice for separating mixtures of fatty acids,and the combination of GC-MS methods

allows the resolution of molecular details,

such as the number and position of double

bonds within the fatty acid molecule. Inmeasuring the mouse macrophage lipidome,Quehenberger et al. (25) reported a stable

isotope dilution GC-MS method capable ofquantitating 30 saturated and unsaturated fatty

acids contained in the mouse macrophage ina single analysis. The methodology employs a

rapid extraction procedure of free fatty acidsfrom the cell medium, followed by a relatively

quick and simple derivatization, permitting

a limit of detection in the femtomole range.GC-MS instruments do not employ an ESI

source, so derivatization of the free fatty acidis required to make it volatile in the chemical

ion source used with this instrument.For the other lipid classes listed in Table 1,

HPLC effluent is coupled directly into the massspectrometer and analyzed employing the var-

ious MS/MS scan modes and the high mass ac-curacy/resolution capabilities described above.

Impressive lipidomic analyses of the subcellu-lar organelles of the mouse macrophage (18, 19)

and human blood plasma (21) have been carried

out.Table 1 lists (in the last three columns) thenumber of lipid molecular species accurately

quantitated in each of these three studies.In comparing the two lipidomics ap-

proaches, it is important to note that structuralinformation can be inferred using the elution

order from the HPLC column, which can aidin the characterization of lipids that cannot be

inferred by mass spectrometric analysis alone.One such case is the occurrence of ether-linked

triglycerides in mixtures of triacylglycerols(41). Another such case involves the SRM scan

mode described above. A method has been

created in which the mass spectrometer cancycle through numerous SRM pairs repeatedly;

it is referred to as multiple reaction monitoring(MRM). Coupling a liquid chromatographic

separation to the mass spectrometer and


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a

b28

24

20

16

Mol% 12

8

4

0

TAG

DAG LPA PA LPS LPE LPC LC

BLC

BP Cer IPC MI

PCSte

rols

M(IP)2

CLPI PIPC CLPGPEPS

650

100

80

60

40

20

0

700 750 800

m/z (amu)

Relat

iveintensity(%)

850 900 950

elo1

elo2

elo3

650

100

80

60

40

20

0

700 750 800

m/z (amu)

Relativeintensity(%)

850 900 950

650

100

80

60

40

20

0

700 750 800

m/z (amu)

Relative

intensity(%)

850 900 950

665.6 (14:014:0 PtdGro)

662.6 (15:015:0 PtdEtn)

680.6 (14:114:1 PtdCho) 812.7 (16:022:6 PtdCho)

840.7 (18:122:4 PtdCho)

N16:0 SM709.6

N18:0 SM737.6

16:018:1

PtdCho766.6

16:020:4PtdCho788.6

T17:1

TAG849.7 TAG

Negative-ion ESI/MS of a mouse myocardial lipid extract

Negative-ion ESI/MS of a mouse myocardial lipid extract in the presence of LiOH

Positive-ion ESI/MS of a mouse myocardial lipid extract in the presence of LiOH

(Doubly charged)cardiolipin

16:018:1 PtdGro747.6

16:022:6 PlsEtn746.6

16:022:6 PtdEtn762.6

18:022:6 PtdEtn792.7

PtdEtn

766.7

PtdSer834.7

PtdIns885.7

PlsEtn774.7

PtdGro665.6

18:018:2 PtdGro773.6

18:022:6 PtdSer

834.7

18:020:4 PtdIns885.7

18:122:4 PtdIns911.7PtdIns792.7

662.6

Internalstandard

Internalstandard

Internalstandard

837.7

(O

HN24:0SM)

BY4741


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employing the MRM mode, the mass spec-

trometer is used as a highly selective and highlysensitive HPLC detector. If a specific molecule

is eluting from the chromatographic columnat any time during the analysis, its specified

MRM pair can be detected and recorded. So-

phisticated methods have been developed thatcan monitor hundreds of lipid mediators, such

as eicosanoids, through their defined MRMpairs in a single analysis lasting only 20 min

(42). Figure 5 is an example where such amethod was employed for measuring temporal

genomics and lipidomics changes in eicosanoidbiosynthesis in mouse macrophages in response

to endotoxin stimulation (43); this allowedinvestigators to determine the flux of metabo-

lites (44). As a tool in constructing such MRMmethods for other lipid classes, a highly valu-

able resource for many hundreds of MS/MS

spectra for lipid standards is freely available athttp://www.lipidmaps.org/data/standards/

index.html.

MASS SPECTROMETRYBASEDSTRUCTURE DETERMINATIONOF NOVEL LIPIDS

Targeted versus UntargetedLipidomics

Since its inception nearly 100 years ago, massspectrometryhasplayedacentralroleinthedis-

covery of new molecules. Technological devel-opments that followed its initial inception, such

as tandem mass spectrometry, high mass ac-curacy and resolution capabilities, various ion-

ization methods, and the online coupling ofHPLC, have served well to increase its utility

in the molecule discovery area. The emergingfield of lipidomics has provided fertile ground

12-HHT

15d-PGJ2

12-PGJ2

15d-PGA2

15d-PGD2

PGB2

TXB2

TXA2

PGA2

PGI2

PGD2

PGH2

PGE2 PGF2

6k-PGF1

6k-PGE1

PGJ211dh-TXB2

11-HETE

TXAS

PGIS

PGDS PGES PGFS

COX

TXDH

Figure 5

Example of a comprehensive lipidomics analysis by separation simplifica(CLASS) approach. A heat map shows the temporal changes of various lmetabolites (prostaglandins), as well as gene expression, in endotoxin-stimulated mouse macrophage cells. Enzymes are shown in red font, andprostaglandin lipid metabolites are shown in blue font. The arrows depisynthesis pathways, including various metabolite intermediates andcorresponding enzymes. Changes are represented as a function of time (right), where rectangles indicate mRNA levels and circles indicate lipidmetabolite levels. Greater intensity of red indicates increasing levels; greintensity of blue indicates decreasing levels; and gray represents no chanlevels relative to unstimulated cells. When enzyme activity can result fromultiple genes, each is represented as a separate line. Redrawn and repri

with permission from Reference 43.

to discover and characterize novel lipid species,

and mass spectrometry has and will continue toplay a vital role in this area.

Lipidomics-based assays can be describedas being targeted, where lipid species to be

monitored are known before commencing theanalysis. An example of such a targeted as-

say is the MRM method described above. Al-though MRM-based assays can offer superb

Figure 4

Examples of shotgun lipidomics. (a) Demonstration of intrasource separation, which is used to minimize ion suppression and imdetection of low-level species. Reprinted with permission from Reference 40. (b) Comparison of the lipid composition of yeast cwild-type (BY4741) cells versus those with a fatty acid elongase gene mutation (elo1, elo2, elo3). Reprinted with permissionReference 20.

http://www.lipidmaps.org/data/standards/index.htmlhttp://www.lipidmaps.org/data/standards/index.htmlhttp://www.lipidmaps.org/data/standards/index.htmlhttp://www.lipidmaps.org/data/standards/index.htmlhttp://www.lipidmaps.org/data/standards/index.html

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sensitivity and are ideal for accurate quantita-

tion,basicallyoneonlyfindswhatoneislookingfor. Lipidomics-based assays that are described

as untargeted are more of an exploratory,qualitative survey of the lipid landscape. Op-

erating the mass spectrometer in the full-scan

mode to search for new mass-to-charge ra-

tio peaks is an option for an untargeted assay,where if a novel lipid species is present, onehopes to detect it without previous knowledge,

that is, assuming the species ionizes efficientlyand the sensitivity of the mass spectrometer is

sufficient. However, once a potentially novellipid is observed, further investigation can be

conducted (e.g., via the MS/MS mode) to con-firm its novelty.

Examples of a few of the novel lipids ob-served recently in untargeted assays are pre-

sented below. Additionally, a protocol for an

untargeted assay that can flag low-level, un-expected novel lipids is described.

Examples of Novel, Unexpected Lipids

The discovery and interest in lipids began well

before the early twentieth century invention ofthe mass spectrometer. Impressively, in 1823,

the French chemist Michel Eugene Chevreulpublished Recherches chimiques sur les corps

gras dorigine animale, which culminated hiswork investigating the structure and properties

of lipids. Chevreul was the first lipid specialist

to discover the concept of fatty acids, includingseveral chemical species such as oleic, butyric,

caproic, and steric fatty acids. He also discov-ered cholesterol and glycerol. Amazingly, even

today, new lipid compounds are being discov-ered, and mass spectrometry is playing a central

role in elucidating their chemical structure.Figure 6 shows several examples of some

of the novel, unexpected lipid species that havebeen discovered in living systems during the

past few years. Employing HPLC and tan-

dem mass spectrometry, the chemical struc-tures were obtained for a number of ether-

linked glycerophospholipids (45); an exampleof these molecules is shown in Figure 6a.

While recently profiling the lipidome of the

mouse brain using mass spectrometry, Gu

et al. (46) discovered a novel family of Nacylphosphoserine derivatives; an example

these molecules is shown in Figure 6b.Althoughit wasobserved nearly 30 years a

that the retina was highly enriched with ve

long-chain polyunsaturated fatty acids (4

more recent work employing both GC-MS aHPLC-ESI-MS/MS (4851) methods has lto a more full characterization of these as gly

erophosphocholine species containing the velong-chain polyunsaturated fatty acids at thes

1 position and docosahexaenoic acid at the snposition (see the example shown in Figure 6

Lastly, the in vivo formation of 22-carbodihomoprostaglandins from a 20-carb

arachidonic acid (AA, 20:4, n-6) precurs

was unexpectedly observed in endotoxstimulated mouse macrophage cells (5

and an example of one of these is shown Figure 6d. A sensitive, untargeted lipidomi

approach, DIMPLES/MS, that was critical this discovery is described below.

Untargeted-Flagged Lipidomics

As mentioned above, the ultrasensitive MR

mode has the capability of monitoring numeous molecular species in a single analysis; how

ever, assumptions have to be made in advanregarding the species that might be present

provide MRM pairs for the detection schem

If the goal of untargeted lipidomics is to coduct a global survey of lipids in a system u

der study, then the concern must be raised to whether biologically significant lipid speci

are being overlooked. Are there possibly uexpected species and hence no available MR

pairs that would be required for their detectio To overcome such a dilemma, a m

spectrometrybased stable isotope labelistrategy coined DIMPLES/MS (52) for diver

isotope metabolic profiling of labeled exog

nous substrates using mass spectrometry wdeveloped andis illustrated in Figure 7. Briefl

in these experiments, mouse macrophage cewere incubated in a medium supplemented wi

deuterium-labeled arachidonic acid (AA-d


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(18:0e/20:4)ether-linked glycerophosphoinositol

(34:6/22:6)glycerophosphocholine

Dihomo-prostaglandin D2

a

b

c

d

(18:0/18:1/16:0)N-acylglycerophosphoserine

HO

HO

HO

HO

OH

OH

OH

OH

OH

OH

NH

H

O

O

O

O

HO

OP

O

O

O

O

O

O

H

O

O O

PO

HON+

O

O

POO

O

O

O

O

O

Figure 6

Examples of a few of the novel species observed recently in untargeted lipidomics mass spectrometrybasedassays (4552).

and then stimulated with endotoxin. Separately,

this was also carried out with cells that were notsupplemented with AA-d8. The medium from

each was extracted and individually analyzed

with the mass spectrometer operated in full-scan mode. Two sets of eicosanoid generation

resulted, one set from endogenous AA andthe other from the supplemented exogenous

AA-d8. This results in a characteristic andobvious doublet pattern or flag, resolvable with

mass spectrometry, allowing for a sensitive and

comprehensive eicosanoid search without anyprevious knowledge or assumptions as to what

species may be present.

As shown in the nonsupplemented sample(Figure 7a), prostaglandin D2 (PGD2) was ob-

served at m/z = 351 (the solid blue trace) as ex-pected for endotoxin-stimulated macrophages.

For the AA-d8-supplemented sample (thedotted red trace, data collected separately and


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AA + d8

PGD2 + d7

dih PGD2 + d7

COX+PGD-synthase

COX+PGD-synthase

D DD D

COOH

DD

D

DDO

HO

DOH

D

DD

DDO

HO

OH

D

COOH

DD DD

AA + d8

Adrenic acid + d8

D DD D

D DD D

D D

COOH

COOH

COOH

DD DD

DD DD

Control (nonsupplemented)

AA-d8 supplemented (40 M)

Two-carbon addition

351

352

353

351 353 355 357 359

m/z (amu)

m/z (amu)

Relativeintensity

(%)

356

357

358

359355

385

384

379

383

386380

387381

379 381 383 385 387

Relative

intensity(%)

a

b

[(PGD2 + d6) H]

[(dih PGD2 + d6) H]

[(dih PGD2 + d7) H]

[(PGD2 + d5) H]

[(PGD2 + d7) H]

[PGD2 H]

[dih PGD2 H]

Figure 7

Example of the diverse isotope metabolic profiling of labeled exogenous substrates using mass spectrometr

(DIMPLES/MS) approach. (a) The characteristic doublet pattern, the observation of prostaglandin D2(PGD2), and deuterium-labeled PGD2 produced by macrophage cells supplemented with deuterium-labearachidonic acid (AA-d8). (b) The unexpected observation of 22-carbon dihomo-prostaglandin D2(dih-PGD2), resulting from the 2-carbon elongation of arachidonic acid (AA). Redrawn and reprinted withpermission from Reference 52. amu, atomic mass unit; COX, cyclooxygenase; m/z, mass-to-charge ratio.The y-axis is percentage of relative intensity, with the top of the y-axis 100%; the numbers (e.g., 351, 352,etc.) above the peaks correspond to their amu location on the x-axis.


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overlaid), a mass offset pattern, not observed

in the nonsupplemented sample, is clearly ob-served, resulting from the action of eicosanoid-

producing enzymes, cyclooxygenase (COX)and PGD-synthase, on the AA-d8 substrate.

Note that, even in the supplemented sample,

some nondeuterated PGD2 is observed, indi-

cating some endogenous AA within the cell re-mains to serve as a substrate.In the above example, the production of

PGD2 was expected and shown for the pur-pose of the DIMPLES/MS demonstration. Us-

ing the DIMPLE/MS strategy, and as shownin Figure 7b, an unexpected and particu-

larly interesting observation was the produc-tion of 22-carbon dihomoprostaglandins, prod-

ucts of adrenic acid (22:4, n-6), resulting fromthe 2-carbon elongation of AA by the stim-

ulated mouse macrophage (52). Although it

had been previously observed that adrenicacid could serve as a COX substrate when

added to cells, resulting in the productionof dihomoprostaglandins (53, 54), the DIM-

PLES/MS strategy revealed its formation denovo from an arachidonate precursor. Even

though this DIMPLES/MS demonstration wasfor AA and prostaglandin production, similar

labeling strategies should prove equally valu-able for other substrates and lipid species.

PROTEIN/ENZYMEASSOCIATION WITH LIPIDSAND MEMBRANES

Peptide Amide Hydrogen/Deuterium Exchange MassSpectrometry

DXMS is a useful technique for studying pro-

tein dynamics and folding, ligand binding, andprotein-protein interactions; it strongly com-

plements and adds to the X-ray crystallogra-phy and nuclear magnetic resonance methods

for the exploration of protein structure (5559).

Recently, DXMS hasproven to be a particularlyvaluable technique for exploring the location

and orientation of proteins and enzymes asso-ciated with individual lipid molecule membrane

phospholipids (6062).

Utilizing DXMS as a tool for studying

protein structure and interactions is based onthe principle of hydrogen exchange with a

solvent. As illustrated in Figure 8a, hydrogenatoms contained on a protein molecule can be

divided into three classes based on their rate of

exchange with an aqueous solvent: those that

hardly ever exchange, those that exchangeextremely quickly, and those whose exchangerate depends on their local environment. As

such, amide hydrogen atom exchange rates ina protein vary depending upon their environ-

ment/location and can be used to investigatethe structural dynamics of the protein.

Basic DXMS methodology is as follows:The protein of interest is incubated in deuter-

ated water for different periods of time, during

which the protein can be subjected to variousadditional substances, probes, or perturbations

to explore how this may influence its wateraccessibility and conformation and, hence,

local amide hydrogen exchange rates. Amidehydrogen atoms that are in the hydrophobic

regions of the protein need to be incubatedlonger to exchange, or may not exchange

at all, whereas those on the exposed surfaceexchange quickly at the start of the incubation.

The deuterium atoms can then be locked inplace or quenched and prevented from further

exchange by lowering the solution pH to 2.5

and temperature to 2C. An HPLC-mass

spectrometry analysis is then conducted that

is similar to a typical bottom-up proteomicsanalysis (63, 64) wherein a protease is used

to digest the protein into its correspondingpeptides, each generally on the order of 5 to

15 amino acids in length. These peptides mayalso be fragmented into smaller pieces in the

mass spectrometer (using the MS/MS scanmode), which helps in their identification and

in the sequencing of the protein. In DXMSexperiments, the instrumentation is often

customized to include a component such as an

online chilled pepsin protease digestion HPLCcolumn, which minimizes deuterium back ex-

changeand helps automate the analysis (6062).The data collected from various deuterium

incubation times are then used to construct


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a

b

O

C

O

O O

C

C C

O

C

O

C

O

CCCH NH NH NHNH

SH

OH

OH

OH

NH2

NH2

H2N

CH2 CH2

CH2

CH2

CH2

CH2 CH2

CH2

CH2

CH2

CH CH CHH

i ii iii

Figure 8

Deuterium exchange mass spectrometry (DXMS) used to investigate protein/enzyme associations with lipi(a) Hydrogen atoms contained on a protein molecule can be divided into three classes based on their rate oexchange with the aqueous solvent. Those attached directly to carbon atoms (blue) hardly ever exchange;those attached to amino acid side chain atoms, the N-terminal amine, and the C-terminal carboxylic acid(green) exchange extremely rapidly; and amide hydrogen atoms (red) have variable exchange rates fromseconds to months depending on the protein conformation and solvent accessibility. (b) Depictions of themembrane interactions for representatives of the three main kinds of phospholipase A2 (PLA2), (i) the sm

secreted sPLA2 (reprinted with permission from Reference 60), (ii) the cytosolic cPLA (reprinted withpermission from Reference 61), and (iii) the calcium-independent iPLA2 (reprinted with permission fromReference 62). Each has a different and distinct interaction with the phospholipid membrane.

maps that indicate which regions of the pro-

tein contain amide hydrogens that exchangequickly,slowly,ornotatall,aswellasthedegree

of surface exposure. This technique has nowbeen used successfully to examine the inter-

action between individual lipid molecules andphospholipids in membrane vesicles to deter-

mine which peptides interact. One limitation to

this approach is that collision-induced dissoci-ation (the usual fragmentation mechanism used

with most mass spectrometers, including triplequadrupole instruments) causes scrambling,

whereby, upon fragmentation, the deuteriumatom changes position within the peptide (65).

Electron transfer dissociation, a more recently

developed fragmentation mechanism, appea

to produce minimal scrambling under the corect experimental conditions (66), suggesting

will indeed be possible to achieve single-residresolution of hydrogen/deuterium exchange r

actions (67). So in the future, even more pr

cise information on lipid conformations whbound to proteins should be possible.

Association of Proteins/Enzymeswith Lipids and Membranes

The use of DXMS to study the interaction proteins with lipids and membranes has be

sparse (68, 69), but this technique has recen


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been applied to several members of the phos-

pholipase A2 (PLA2) enzyme superfamily (6062) interacting with lipid molecular species in

their active sites as well as with phospholipidvesicle membranes. One of the major phos-

pholipase A2s, the group IVA cytosolic PLA2(cPLA2) (61), is very important in cell sig-

naling and in the release of AA, the precur-sor of numerous inflammatory mediators (e.g.,prostaglandins).DXMSstudieswith two potent

and specific reversible lipid inhibitorssuggestedspecific docking modalities, each in a different

specific location at or near the catalytic site(70).When these inhibitors were docked and then

subjected to molecular dynamics calculations,their precise binding conformations were fur-

ther defined giving a precise picture of the pro-teins interaction with these two lipid inhibitors

and their bound conformations. One of these is

a substrate analog, so it can provide an excellentpicture of the conformation of a phospholipid

molecular species bound in the active site of thisenzyme (70).

When this same cPLA2 is presented witha phospholipid vesicle membrane, changes in

several peptide deuterium exchange rates wereobserved, suggesting the peptidic locations

of interaction with the vesicle. Both the C2domain and the / hydrolase fold domain

showed such changes, leading to the conclu-sion that the large cap region, which includes

a lid normally covering the active site, under-

goes a broad interaction with the membranephospholipids (61). Each hasa differentanddis-

tinct interaction with the phospholipid mem-brane. Although the 85-kDa cPLA2 interacts

with both its C2 domain and its large cap re-gion, in contrast, the smaller 13-kDa secreted

sPLA2 inserts a limited number of amino acidside chains into the phospholipid surface (60).

In further contrast, the 85-kDa iPLA2, whichhas both a large ankyrin repeat domain (not

shown in the figure) and an / hydrolase fold

domain, contains an exposed helix, which in-serts into the membrane with little or no effect

on the active site (62). With the further refine-ments discussedabove, DXMS hasthepotential

for providing even more precise pictures of the

conformation of lipids when interacting with

proteins in active sites and in membranes.

CELL/TISSUE IMAGING OFLIPIDS BY MASS SPECTROMETRY

Localization of Lipids in Tissues

and Cells

The mass spectrometrybased lipidomics anal-yses described above have greatly advanced our

knowledge regarding the diversity and numberof unique molecular lipid species contained in

biological systems. Knowledge of the subcellu-lar localization of individual lipids can provide

additional detailed insight into lipid functions,

such as those involved in disease and cell death.Employing differential centrifugation for sub-

cellular organelle isolation, Andreyev et al. (18)recently provided the first mapping of the sub-

cellular mouse macrophage lipidome in basaland activated states, focusing on the major lipid

species and the major organelles. A total of 229individual/isobaric species were identified, and

these are shown according to lipid class in Ta-ble 1. Although highly impressive, it is possible

that this isolation technique might chemically

alter the lipids under study or their location.Additionally, if one is studying tissue samples,

the lipid extraction procedure clearly destroysinformation regarding the location of the lipid

within the tissue. Certainly, carrying out a massspectrometric lipid analysis of tissue samples in

situ and, ultimately, individual cells might avoidsuch pitfalls andprovide us with an unperturbed

picture of the cellular and subcellular lipidome.

Matrix-Assisted Laser DesorptionIonization-Imaging MassSpectrometry

A relatively new technique, pioneered by

Caprioli and colleagues (71) within the past

decade, is imaging mass spectrometry (IMS),which provides spatially resolved ion inten-

sity maps corresponding to the mass-to-chargeratio of intact molecular species at specific

locations in a prepared tissue sample. A most


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common mode of IMS employs an ultraviolet

(UV) laser to form ions from molecules con-tained in a tissue sample that is coated with

a UV-absorbing matrix, and as such, this isknown as matrix-assisted laser desorption ion-

ization(MALDI). MALDI waspioneeredin the

1980s by Karas et al. (72) and Tanaka et al.

(73); Koichi Tanaka shared the 2002 NobelPrize in Chemistry for this contribution (asnotedabove,JohnFennwasawardedpartofthis

same Nobel Prize for his contribution to ESI).Briefly, MALDI-IMS involves preparing a thin

(10-m thick) slice of tissue sample, whichis mounted onto a small plate (MALDI target,

approximately 3 cm 3 cm) and then coatedwith a film of a UV-absorbing compound (ma-

trix), such as dihydroxybenzoic acid. Firing asmall UV laser spot onto the matrix/tissue sam-

ple generates an energetic plasma, resulting in

the formation of both positive and negativebiomolecular ions from the tissue, which are

ejected from the target surface and coupled di-rectly into the entrance of a mass spectrometer.

As with ESI, MALDI is a soft ionization tech-nique, leaving the molecule whole and unfrag-

mented in the ionization process. A mass-to-charge ratio spectrum is recorded for each X/Y

grid coordinate on the target the MALDI laseris focused upon.

The prepared target is placed atop amechanical stage, which is programmed to

be moved incrementally such that an array of

mass-to-charge ratio spectra are recorded forthe entire tissue sample. Software is then used

to generate mass-to-charge ratio images as afunction of the X/Y grid location for the entire

tissue sample. While data is recorded for allmass-to-charge ratio values within the specified

mass scan range, software permits visualizationof designated (extracted) values, enabling lipid

species-specific images and comparisons. Cur-rent laser technology permits MALDI-IMS to

have a lateral resolution on the order of 25

100 m. Owing to its fast duty cycle, which al-lows it to be compatible with a high-repetition-

rate MALDI laser, and its high mass accuracycapability, the TOF mass spectrometry is the

instrument most frequently used for imaging.

Most of the early MALDI-IMS work involv

peptide and protein imaging in tissue sampl(71, 7477); however, recently, impressi

results from imaging lipids have been report(7882). Figure 9a is an extractedion image f

m/z = 772.5 from a mouse brain slice that w

obtained using a TOF mass spectrometer sc

range of m/z 500 to 900. The 16:0/16:0-PCK+ (potassium cation adduct) species hascorresponding mass of 772.5 Da. Using the

appropriate corresponding accurate masssimilar extracted ion images are possible f

other gylcerophospholipid species collected this data set. Although such an image sugges

that regional differences in concentrations fspecific molecular species are present, add

tional work is still needed to determine if ioabundance truly reflects local concentratio

(81). Observed signal intensities may be skew

owing to local intracellular concentrations sodium and potassium, local environments

tissue slices, as well as other factors.The specialized MS/MS scan modes, whi

are very useful for monitoring various lipclasses (described above), using the trip

quadrupole mass spectrometer can alwabe utilized with MALDI-IMS. The tande

quadrupole-TOF instrument is a particularuseful instrument for such work, as the first tw

sectors of this instrument are the same as thtriple quadrupole and capable of the many sc

modes shown in Figure 3. However, the thi

sector is a TOF unit with high mass accuracapabilities.

Future Directions in ImagingMass Spectrometry

Although IMS is still in its relative infancit has already provided a highly valuable to

to the lipidomics investigational toolchest, an we can expect its contributions and capabi

ties will only continue to grow in the futur

One area that is aggressively being pushed foward is the ability to conduct single-cell, intr

cellular imaging. This is not possible with tcurrent lateral resolution capabilities availab

with MALDI-IMS (25100 m); however, t


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use of focused ion beam clusters as a means of

initiating ionization is showing much promise.Using highly focused ion beam clusters, such

as Buckminsterfullerenes (C60 clusters), lateralresolutions on the nanometer scale have been

reported (83, 84), putting this technique well

within the range required to allow single-cellimaging. As lateralresolution is improved,how-

ever, the amount of ablated imaging target ma-terial decreases; thus, improvements in mass

spectrometer sensitivity need to keep step. Ad-ditionally, as lateralresolutionimproves so does

the size of the collected data set. This will re-quire concomitant improvements in data pro-

cessing and storage.Another area of development in IMS in-

volves coupling an ion mobility spectrometerbetween the ionization source (MALDI or ion

beam cluster) and the mass spectrometer. Ion

mobility spectrometry separates analyte ions onthe basis of their ion-neutral collision cross sec-

tion (85). This is accomplished by having ions,upon slight acceleration, drift through a tube

maintained at 35 torr of helium gas beforebeing directed into a coupled mass spectrom-

eter. Ions having the same mass-to-charge ra-tio yet different three-dimensional shapes, and

Figure 9

Examples of matrix-assisted laser desorptionionization imaging mass spectrometry used toinvestigate lipids in tissue samples. (a) Extracted ionimage for m/z = 772.5 from a mouse brain slice.The mass of 772.5 Da corresponds to the16:0/16:0-PC + K+ (potassium cation adduct)species. (b) Images from a section of mouse kidney.Using MS/MS scan mode and high mass accuracy toconfirm identity, cholesterol (i), 16:0/18:2-PC (ii),40:6-PC (iii), and 16:0/20:4/18:1-TAG (iv) weresome of the lipid species observed. Panels a and breprinted with permission from Reference 81.(c) Example of an ion mobility-mass spectrometry(IM-MS) two-dimensional plot for a nominallyisobaric peptide (RPPGGFSP) and lipid (32:4-PC).The mass-to-charge ratio (m/z) is plotted on thex-axis, and ion mobility drift time is plotted on they-axis. The mass-to-charge ratio signals overlap andcannot be resolved; however, they clearly havedifferent ion mobility drift times. Reprinted withpermission from Reference 88.

c540

520

500

480

Arrivaltimedistribution(s)

460

440

420

400730 740 750 760

[RPPGFSP + H]+

[Phosphatidylcholine 34:2 + H]+

m/z (amu)

770 780 790

b i ii

iii iv

a

Cerebral cort

Corpus callos

Hippocampu

Thalamus

Hypothalam

StriatumIntensity

m/z 772.5

1 mm

m/z 796.6m/z 369.3

m/z 879.7m/z 862.6


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therefore different collision cross sections, have

different drift times through the helium gas andcan be resolved. For example, different molecu-

lar classes exhibit significant differences in theircollision cross section at a given mass-to-charge

ratio, e.g., oligonucleotides/carbohydrates

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