mass spectrometry of lipids and glycoconjugatesj chromatogr b analyt technol biomed life sci. 2009,...

1

Mass Spectrometry of Lipids and Mass Spectrometry of Lipids and GlycoconjugatesGlycoconjugatesGlycoconjugatesGlycoconjugates

Catherine E. CostelloCatherine E. Costello

BI 793 Lecture BI 793 Lecture 1111

April April 13, 201013, 2010

Some types of lipids in biological systemsSome types of lipids in biological systems

Membrane Lipids:Membrane Lipids: Fatty acids, steroids, glycolipids,Fatty acids, steroids, glycolipids,lipopolysaccharides (LPS) glycerophospholipids lipid raftslipopolysaccharides (LPS) glycerophospholipids lipid raftslipopolysaccharides (LPS), glycerophospholipids, lipid rafts, lipopolysaccharides (LPS), glycerophospholipids, lipid rafts, caveolae caveolae ProteinProtein-- and carbohydrateand carbohydrate--bound lipids: bound lipids: anchorsanchorsSphingolipids:Sphingolipids: ceramides, sphingomyelinceramides, sphingomyelinGlycosphingolipids:Glycosphingolipids: cerebrosides, gangliosidescerebrosides, gangliosidesEicosanoids:Eicosanoids: prostaglandins, leukotrienes, lipoxins, prostaglandins, leukotrienes, lipoxins, isoprostanesisoprostanesFatFat--soluble Vitamins:soluble Vitamins: A, D, E, KA, D, E, KCD1 lipid antigen presentationCD1 lipid antigen presentation

2

R2-COO-CH

CH2-OOC-R1

CH2-O-P-O-(CH2)2-N(CH3)3

O

O

Phosphatidylcholine

R-CONH-CH

HO-CH-CH=CH-(CH2)12-CH3

CH2-O-P-O-(CH2)2-N(CH3)3

O

O

Sphingomyelin

CH3

CH3

HO

Structures of common lipidsStructures of common lipids

Phosphatidylcholine

R2-COO-CH

CH2-OOC-R1

CH2-O-P-O-(CH2)2-NH3

O

O

Phosphatidylethanolamine

CH2 OOC R1

Sphingomyelin

R-CONH-CH


CH2-O-Glc

Glucosylcerebroside

HO CH CH=CH (CH )12 CH3

Cholesterol

R2-COO-CH

Triglyceride

R1-COO-CH2

R3-COO-CH2

R2-COO-CH

CH2-OOC-R1

CH2-O-P

O

O

Phosphatidylinositol

O

OH OH

OH

OHHO

Sia-(2,3)-Gal-(1,4)-Glc-(1,1)-O-CH2

Gal-β(1-3)-GalNAc

(1,4)

R-CONH-CH


Ganglioside GM1

LipidMaps LipidMaps classification classification systemsystem

J. Lipid Res., J. Lipid Res., 20052005, , 4646, 839, 839--872872

3

Some roles of lipidsSome roles of lipids

Membrane Lipids:Membrane Lipids: structural components, organization of structural components, organization of membrane proteinsmembrane proteins etcetc (lipid rafts) regulation of cell(lipid rafts) regulation of cell--cellcellmembrane proteins membrane proteins etc.etc. (lipid rafts), regulation of cell(lipid rafts), regulation of cell cell cell and celland cell--ligand interactionsligand interactionsSphingolipids:Sphingolipids: structural components, signalling, second structural components, signalling, second messengers, biosynthetic intermediatesmessengers, biosynthetic intermediatesProtein and CarbohydrateProtein and Carbohydrate--bound Lipids:bound Lipids: anchors for anchors for biopolymersbiopolymersProstaglandins, eicosanoids:Prostaglandins, eicosanoids: signallingsignallingFat soluble vitamins, tissue lipids:Fat soluble vitamins, tissue lipids: free radical traps, free radical traps, response to oxidative stress, energy storesresponse to oxidative stress, energy storesCD1 lipid antigen presentation: CD1 lipid antigen presentation: immune system activationimmune system activation

Lipids in BiologyLipids in Biology

Websites/program to watch:Websites/program to watch:

www.lipidmaps.orgwww.lipidmaps.orgLIPID MLIPID Metabolitesetabolites AAndnd PPathways athways SStrategytrategy

also:also:www.cyberlipid.orgwww.cyberlipid.org

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LipidMaps tools for LipidMaps tools for mass spectrometrymass spectrometry

www.lipidmaps.orgwww.lipidmaps.org

EI MS of isomeric C14 hydrocarbonsEI MS of isomeric C14 hydrocarbons

n-HexadecaneCH3(CH2)14CH3

MW 226

C5

C4C3

C2

5-Methylpentadecane

CH3(CH2)3 CH (CH2)9CH3

CH

C7 C8 C9 C10

C62

C11 C12

C13

C14 M

C16

C3

m/z

M+.

CH3

57 169 85 141

C4

C5

C6

C7 C8 C9 C10

C12

C16

MM-15

m/z

M+.

5

EI MS of methyl caprylateEI MS of methyl caprylate

Methyl caprylate

CH3(CH2)6COOCH3

MW 158

m/z 158 (M) 100.00

m/z 159 (M+1) 12.90

m/z 160 (M+2) 1.00

m/z

M-31M+.

CID MS of [MCID MS of [M--H]H]--, carboxylate , carboxylate anions derived from (A) 13anions derived from (A) 13--oxotetracosanoic acid (oxotetracosanoic acid (m/zm/z 381) 381) and (B) 13and (B) 13--oxotetracosanoic oxotetracosanoic acidacid 12 12 14 1412 12 14 14 dd ((m/zm/z 385)385)acidacid--12,12,14,1412,12,14,14--dd44 ((m/zm/z 385)385)

(C) Fragmentation of [M(Li)+Li](C) Fragmentation of [M(Li)+Li]++

of 13of 13--oxooxo--octadecanoic acid octadecanoic acid ((m/zm/z 310)310)

C

6

HighHigh-- and lowand low--energy CID MS fragmentation of the energy CID MS fragmentation of the [M+H][M+H]++ m/zm/z 574 in the positive574 in the positive--ion ESI mass spectra of ion ESI mass spectra of the glutathione conjugates of estrone and estradiolthe glutathione conjugates of estrone and estradiol

Ramanathan et al., JASMS, 9, 612 (1998)

HighHigh--energy CID fragmentation of [Menergy CID fragmentation of [M--H]H]-- m/zm/z 657 in the 657 in the negativenegative--ion FAB mass spectrum of sulfonated and ion FAB mass spectrum of sulfonated and glucuronidated double conjugate of 24glucuronidated double conjugate of 24--hydroxy cholesterol hydroxy cholesterol

Meng et al., J Lipid Res., 38, 926 (1997)

7

GlycerophospholipidsGlycerophospholipids

1,21,2--diacyl 1diacyl 1--OO--alkyl,2alkyl,2--acyl plasmalogensacyl plasmalogens

R1

1,21,2 diacyl 1diacyl 1 OO alkyl,2alkyl,2 acyl plasmalogensacyl plasmalogensRR11, R, R22, R’ = alkyl chain; lysophospholipid 2, R’ = alkyl chain; lysophospholipid 2--OHOH

MonoMono--, di, di-- and triacylglyceridesand triacylglycerides

Glycerophospholipid polar head Glycerophospholipid polar head groups (X)groups (X)

glycerophosphoserine (GPS)glycerophosphoserine (GPS)-O glycerophosphoserine (GPS)glycerophosphoserine (GPS)

glycerophosphoethanolamine (GPE)glycerophosphoethanolamine (GPE)

glycerophosphocholine (GPC)glycerophosphocholine (GPC)

-O

-O

glycerophosphoinositol (GPI)glycerophosphoinositol (GPI)-O

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Characteristic fragment, neutral losses for Characteristic fragment, neutral losses for glycerophospholipid polar head groups (X)glycerophospholipid polar head groups (X)

GPC GPC GPEGPE

GPSGPS GPIGPI

Characterization of a lipid mixture by nanospray MSCharacterization of a lipid mixture by nanospray MS

Instrument: Finnigan-MAT TSQ 7000 triple quadrupole MS

Nanospray MS

Other useful scans (not from this ref.):PI pos.: NL of 260 Cholesterols pos.: PC of 369Fatty acids neg.: PC 255, 279, 281, 303 etc.Sugars pos.: PC 163, 204, 292 etc.

MeOH/CHCl3 2:1, in pos. ion mode 1% HOAc

B. Brügger, G. Erben, R. Sandhoff, F. T. Wieland, and W. D. Lehmann (1997). Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc.Nat.Acad.Sci.USA 94, 2339-2344.

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LCLC--MS of lipidsMS of lipids

Why LC?Why LC?- Quantification of minor components (signal suppression)- Sample cleanup: getting rid of cluster formers etc.- Retention time helps to characterize a compound.

Why MS? (instead of light scattering, UV etc)Even simple MS allows an immediate impression of the-Even simple MS allows an immediate impression of the

molecular species involved, isomers excepted.

U. Sommer, H. Herscovitz, F. K. Welty, and C. E. Costello., J. Lipid Res., 2006, 47, 804-814

SE: sterol esters, S: sterols, DAG: diacylglycerols, MGDG: monogalactosyldiglycerides, SG: sterol glycosides, CERE: cerebrosides, DGDG di l ldi l idDGDG: digalactosyldiglycerides, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PC: phosphatidylcholine

Column: YMC PVA-Sil (250 x 4.6 mm, 3 µm from Hichrom), the phase is prepared by bonding a layer of polymerized vinyl alcohol to silica.Ternary HPLC pump (40 min @ 1 ml/min, 10 min reequilibration time)Evaporative light-scattering detectorp g g

Solvent A: isooctane/methyl tert-butyl ether (98/2, v/v)Solvent B: isopropanol/acetonitrile/chloroform/acetic acid (84/8/8/0.025, v/v)Solvent C: isopropanol/water/triethylamine (50/50/0.2, v/v)Isooctane may be replaced by isohexane (for safety reasons), with similar results.

(from www.cyberlipid.org, after W.W.Christie et al. (1995). J High Resol Chromatogr 18, 97.)

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Lipid QuantificationLipid Quantification

Ion intensity depends on:

-Ionization efficiency of head groups

-Fatty acid length and saturation

-Suppression

-Solvent composition (small changes)changes)

-Instrument settings (actual settings can vary from day to day!)

B Brügger, G Erben, R Sandhoff, FT Wieland, WD Lehmann, PNAS, 1997, 94, 2339-2344

PC Lipids in B100 (WT) and B67 LDLsPC Lipids in B100 (WT) and B67 LDLs

(Sommer et al.)

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Neutral Lipids in RAW 264.7 cellsNeutral Lipids in RAW 264.7 cells

TLC separation of (A) isooctane-EtOAc extract of neutral lipids (B) Bligh-Dyer extract of total lipids

(Hutchins et al.)

(A) Normal-phase LC separation and MS detection as precursors of m/z 369.3 or as total ion current of neutral lipids from RAW cells. (B)-(D) Overlaid mass chromatograms of selected [M+NH4]+.

extract of total lipids

Neutral Lipids in RAW 264.7 cellsNeutral Lipids in RAW 264.7 cells

(Hutchins et al.)

Summed positive-ion MS data recorded for precursors of m/z 369.3 or as total ion current of neutral lipids from RAW cells. (A) CE fraction (B) MeDAG fraction (C) TAG fraction (D) DAG fraction. Selected [M+NH4]+ of samples and internal standards.

12

Location of sites of unsaturation in lipidsLocation of sites of unsaturation in lipids

Ozone-Induced Dissociation: Elucidation of Double Bond Position within M S l d Li id IMass-Selected Lipid IonsMC Thomas, TW Mitchell, DG Harman, JM Deeley, JR Nealon, SJ Blanksby, Anal. Chem., 2008, 80, 303–311.

Identification of double bond position in lipids. From GC to OzID.TW Mitchell, H Pham, MC Thomas, SJ Blanksby, J Chromatogr B Analyt TechnolBiomed Life Sci. 2009, 877, 2722-2735.

Online ozonolysis methods for the determination of double bond position in unsaturated lipids. MC Thomas, TW Mitchell, SJ Blanksby. Methods Mol Biol. 2009, 579, 413-441.

OzID location of sites of unsaturation in lipidsOzID location of sites of unsaturation in lipids

MC Thomas, TW. Mitchell, DG Harman, JM Deeley, JR Nealon, SJ Blanksby, Anal. Chem., 2008, 80, 303–311.

13


TW Mitchell, H Pham, MC Thomas, SJ Blanksby, J Chromatogr B Analyt Technol Biomed Life Sci. 2009, 877, 2722-2735.

OzOz--ESI for location of sites of unsaturation in lipidsESI for location of sites of unsaturation in lipids

ESIESI


14


The OzESI MS spectrum of a 1 M methanolicThe OzESI–MS spectrum of a 1 M methanolic solution of (a) PC(18:1(9 Z)/18:1(9 Z)) and (b) PC(18:1(6 Z)/18:1(6 Z)). Both spectra are recorded as m/z 184 precursor ion scans and thus show only the [M+H] + molecular ionand corresponding chemically induced fragment ions. The symbols ■ and ●identify the ozonolysis product ions as methoxyhydroperoxides and aldehydes,respectively (reproduced from ref. [103] with


espect e y ( ep oduced o e [ 03] tpermission).

Lipids analyzed by DESI directly from samplesLipids analyzed by DESI directly from samples

Mass spectrometric imaging of lipids using desorption electrospray ionization.

Dill AL, Ifa DR, Manicke NE, Ouyang Z, Cooks RG. J Chromatogr B Analyt Technol Biomed Life Sci. 2009, 877, 2883-2889.

courtesy of R. G. Cooks, Purdue University

15





16



EicosanoidsEicosanoids

17

ESIESI--CID MS of anions, CID MS of anions, m/zm/z 337, from isomeric 337, from isomeric dihydroxyeicosatetraenoic acidsdihydroxyeicosatetraenoic acids

P Wheelan et al., JASMS, 1996, 7, 140

Fragmentation of anions, Fragmentation of anions, m/zm/z 337, from isomeric 337, from isomeric dihydroxyeicosatetraenoic acidsdihydroxyeicosatetraenoic acids

P Wheelan et al., JASMS, 1996, 7 140

18

ESIESI--CID MS of leukotriene ECID MS of leukotriene E44, [M, [M--H]H]-- m/zm/z 438438

RC Murphy et al., Chem. Rev., 2001, 101, 479

m/z

Fragmentation of leukotriene EFragmentation of leukotriene E44, [M, [M--H]H]-- m/zm/z 438438

RC Murphy et al., Chem. Rev., 2001, 101, 479

19

Lipids and oxidative stressLipids and oxidative stress

Mitochondrial fatty acid metabolismMitochondrial fatty acid metabolism

20

Application of acylcarnitine and amino acid Application of acylcarnitine and amino acid analysis (ca. 50 metabolites)analysis (ca. 50 metabolites)

l i i (H C) N CH CH(OR)CH COO

Newborn screening Newborn screening

acylcarnitine = (H3C)3N+CH2CH(OR)CH2COO-

R = CH3(CH2)nCO-

–– for ~30 metabolic disordersfor ~30 metabolic disorders

D. Millington et al., Duke Univ. Medical Center

Fatty acid metabolism overviewFatty acid metabolism overview(courtesy D. Millington)

Mitochondrial fatty acid Mitochondrial fatty acid --oxidation (FAO) is a physiological oxidation (FAO) is a physiological response to tissue energy depletion when fasting, during response to tissue energy depletion when fasting, during febrile illness, and increased muscular activityfebrile illness, and increased muscular activity

In the liver, FAO fuels synthesis of ketone bodies, used as an In the liver, FAO fuels synthesis of ketone bodies, used as an alternative energy source for the brain (especially) and other alternative energy source for the brain (especially) and other extrahepatic organs when glucose reserves are exhausted extrahepatic organs when glucose reserves are exhausted (after only a few hours in neonates)(after only a few hours in neonates)

FAO is the primary energy source for the heart muscle at all FAO is the primary energy source for the heart muscle at all timestimes

21

Fatty acid metabolism overview Fatty acid metabolism overview -- continuedcontinued

More than 20 diseases of the FAO pathway have beenMore than 20 diseases of the FAO pathway have been More than 20 diseases of the FAO pathway have been More than 20 diseases of the FAO pathway have been described to date described to date –– all are of autosomal recessive all are of autosomal recessive inheritance; most are treatableinheritance; most are treatable

The clinical manifestations are diverse, and can include The clinical manifestations are diverse, and can include hypoglycemia, vomiting, lethargy, coma, encephalopathy hypoglycemia, vomiting, lethargy, coma, encephalopathy (Reyes(Reyes--like syndrome), sudden unexpected death, liver like syndrome), sudden unexpected death, liver ( y( y y ) py ) pfailure, cardiomyopathy, respiratory distress, skeletal failure, cardiomyopathy, respiratory distress, skeletal myopathy, myoglobinuria, pregnancy complications myopathy, myoglobinuria, pregnancy complications

The symptoms are often episodicThe symptoms are often episodic

Basis of a recessive metabolic diseaseBasis of a recessive metabolic disease

Child inherits mutant Precursor

gene from both parents

(Pathway intermediates)

Missing or defective enzyme BLOCK

(Normal products)

(in blood and urine)

Abnormal metabolites

(courtesy D. Millington)

22

Underlying enzyme deficiency blocks a unique Underlying enzyme deficiency blocks a unique biochemical pathwaybiochemical pathway

Precursors


-A deficiency of the products of the missing enzyme

X (Products)Missingenzyme

Substrate

Precursors

Toxic products

-Accumulation of a toxic substrate behind the block

-Effects of the accumulated substrates on other pathways

(Deficientdownstreamproducts)

enzymeToxic products

Diagnostic testing for FOA disordersDiagnostic testing for FOA disorders(courtesy D. Millington)

The most useful test is the acylcarnitine profile (plasma or whole blood)or whole blood)

Free and total carnitine in plasma should be analyzed at the same time

Urine organic acids analysis is helpful in diagnosis of some FAO disorders

In vitro testing (skin fibroblasts) and/or molecular testing is often necessary for confirmation

23

Fragmentation of acylcarnitines in Fragmentation of acylcarnitines in MSMS--MS (c) as MS (c) as nn--butyl estersbutyl esters

CRO

N+ CH2(H3C)3 CH CH C

O HO

O C4H8 H

CID

MH+

- N(CH3)3

CID - RCO2H

+CH2 CH CH CO2H m/z 85

- C4H8


Analysis of acylcarnitines by MS/MS Analysis of acylcarnitines by MS/MS in newborn’s blood spotin newborn’s blood spot

C2

C18:1**

Internal standards marked by *

NORMAL

*

C3 C4C4DC

C16 C18

*

**


24

LCHAD or TFP deficiency (metabolic stress)LCHAD or TFP deficiency (metabolic stress)

C16 C16-OH*

C2

C12

C14:1

C18-OH

C18:1

C18:1-OH*

*

C14:1-OH

Reduced C2

**

C10


MS/MS newborn screening in North Carolina: MS/MS newborn screening in North Carolina: 5 yr Summary5 yr Summary

Total screened = 635,168 (7/28/97 to 12/31/02)Total screened = 635,168 (7/28/97 to 12/31/02)Confirmed diagnoses after abnormal screen:Confirmed diagnoses after abnormal screen:Confirmed diagnoses after abnormal screen:Confirmed diagnoses after abnormal screen:

Fatty acid oxidation disordersFatty acid oxidation disorders 6262MCADD (48)MCADD (48)

Organic acidemiasOrganic acidemias 373733--methylcrotonylglycinuria (13)methylcrotonylglycinuria (13)

A i id di dA i id di d 4343 Amino acid disordersAmino acid disorders 4343PKU/hyperphenylalaninemia (30) __PKU/hyperphenylalaninemia (30) ____

Total = 142Total = 142

Overall Incidence: 1:4,473 Overall Incidence: 1:4,473 (D. Frazier, (D. Frazier, UNC)UNC)


25

2 5

3

dic

al

M)

Myocardial ischemia increases free radical generationMyocardial ischemia increases free radical generation

0

0.5

1

1.5

2

2.5

An

iso

tro

pic

fre

e ra

dco

nce

ntr

atio

n (

µM

C t l 10’ I h iControl 10’ Ischemia

J. L. Zweier et al. Proc Natl Acad Sci USA 1987, 84, 1404-1407

Free radical generation during myocardial ischemia. EPR spectra ofcontrol and ischemic heart (inset).

Ischemia and reperfusion cause oxidative stressIschemia and reperfusion cause oxidative stress

V. Palace, J Mol Cell Cardiol 1999 31:193-202

G. Paradies, Free Radic Biol Med. 1999 27, 42-50

26

ROS are generated in the heart during ischemiaROS are generated in the heart during ischemia

O2

One-electron

reductionResidual O2˙- Lipid peroxidation

B. G. Hill, BUSM OPTM in Cardiovascular Disease Symposium, October 2004

Lipid peroxidation generates aldehydesLipid peroxidation generates aldehydes

PROPOSED MECHANISMS• Esterbauer Dioxetane Mechanism• Hydroperoxy Dihydropyran Mechanismy p y y py• Epoxy Hydroperoxide Mechanism• Hydroxyhydroperoxide Mechanims• Enzymatic Pathway• Peroxycyclization-Dioxetane Fragmentation

27

Global ischemia induces HNE modification of Global ischemia induces HNE modification of proteins in the heartproteins in the heart

40’ 30’ 30’ Ischemia 100’Perfusion Ischemia 60’ Reperfusion Perfusion

< 70 kDa

< 65

< 50

< 45

Specific proteins are modified toa greater extent during ischemiavs. ischemia-reperfusion.

Myocardial proteins were separated by SDS-PAGE followed by Western blot (WB) analysiswith anti-protein-HNE antibodies.


## ProteinProtein MW MW (Da)(Da)

pIpI # # Peptides Peptides MatchedMatched

CoverageCoverage Mowse Mowse Score*Score*

Accession # Accession # (gi(gi#)#)

MALDIMALDI--TOF/MS identification of proteins displaying positive TOF/MS identification of proteins displaying positive immunoreactivity with antiimmunoreactivity with anti--proteinprotein--HNE antibodiesHNE antibodies

11 ATP ATP synthase synthase

subunitsubunit

5117151171 4.924.92 1212 34%34% 213213 13747151374715

22 HSP 60HSP 60 5806158061 5.355.35 1111 27%27% 185185 13342841334284

33 MDH2MDH2 3608936089 8.928.92 66 20%20% 7373 1359214513592145

44 VDAC1VDAC1 3085130851 8.628.62 99 46%46% 171171 67559636755963

55 LDH BLDH B 3687436874 5.705.70 88 27%27% 103103 69811466981146

66 EnolaseEnolase 4727347273 7.597.59 88 20%20% 9898 69788116978811

77 Creatine Creatine kinasekinase

4322043220 6.586.58 1010 30%30% 167167 69786616978661


28

C5H11 CH

CH

CH H

OOH

C

4-hydroxy-trans-2-nonanoate (HNA)

C5H11 CH

CH

CH OH

OOH

C

4-hydroxy-trans-2-nonenal (HNE)

H H H H

C5H11 CH

CH

CH H

OHOH

C

Dihydroxynonenone (DHN)GST

C5H11 CH

C CH H

OOH

C

2

H

GS

C5H11 CH

C CH H

OHOH

C

2

H

GS

GS-4-hydroxynonenal (GS-HNE) GS-dihydroxynonenone (GS-DHN)

AR


GlycoconjugatesGlycoconjugates

29

Information available from MS Information available from MS analysis of glycoconjugatesanalysis of glycoconjugates

Molecular weight distributionMolecular weight distribution

Carbohydrate sequence(s)Carbohydrate sequence(s)

Linkage analysis Linkage analysis

Aglycon structureAglycon structureg yg y

Unusual modificationsUnusual modifications

Mixture analysisMixture analysis

Sphingolipids and Sphingolipids and GlycosphingolipidsGlycosphingolipidsGlycosphingolipidsGlycosphingolipids

OH|

R- O-CH2CHCHCH=CH(CH2)nCH3

||NHCO(CH2)mCH3

30

Structures of the gangliosidesStructures of the gangliosides

GM1GD2 GD1b GD1 GT1bGM2

Structures of the Headgroups

GQ1bGQ1

GM1GD1a

CeramideO

HO

O

AcHN

O

HO

O

OH

O

O

HOOC

HOAcHN

HOO H O H

O H

HO

GM2

GD2

GM1GD2 GD1b GD1a GT1bGM2

β-D-Glc α-D-NeuNAcβ-D-Gal β-D-GalNAc

GQ1bGQ1c

OOHO

O H

O

OO

O H

AcHNO

O

HOOC

HO

OO H

O H

HN

OHO

O

HOOC

HOAcHN

HO

O HO H

O H

HO

HOAcHN

( )m

( )n

m = 0, 1, 2n = 1, 2

GD1b

GT1b

100

439.4

x 3

x 1

[M H]-

[M3-H]-

1882.3

QQ--oTOF ESIoTOF ESI--MS of gangliosides from human granulocytesMS of gangliosides from human granulocytes

%

[M4-2H]2-

377.4

[M4-H]-

1992.3

[M1-H]

1517.2

[M3-2H]2-

940.5

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 20000

1816.2

1270.0

1215.0

1013.9

[ 4 ]

995.5

907.9

510.7

[M2-H]-

1627.3

W. Metelmann, J. Müthing and J. Peter-Katalinic, RCMS, 14, 543 (2000)

31

O

OH

HO

CH2OH

O

OH

CH2OH

OH

O CH2

NH

CO

OH

O

NAc

CH2OH

OH

O

OH

HO

CH2OH

ONH

OH

COOHCH

3C

O

O

NAc

CH2OH

OH

O

OH

HO

CH2

OH

O

O

O

O

O

O

OH

OH

OH Y1Y2Y3 Y0Y4Y5

Y6

B2 B3 B5B4

B1

C1

C2 C3 C4

100

Negative ESI-MS/MS of a ganglioside from

x 4

Y 6 /Y 2729.4

Hex-HexNAc364.2

C 1308.0

B 1290.2

% Y 0646.7

Z 0628.3

B 3655.3 C 3

672.3

Z 5 /2,5 A 6 -2H +

653.2

Z 5 /0,2 A 6 -2H +

669.2Y 4 /Y 0687.4

of a ganglioside from human granulocytes

x 1

[M-H] -

1992.3Y 61701.2

Y 51539.0

Y 41336.0

Y 31174.0

Y 2970.9

B 51020.5

C 4833.4

Y 1808.8

C 2468.3

Z 51521.0

Z 31155.9

620 630 640 650 660 670 680 690 7000

Y 4/Y 2-H 2O438.5

Y 0646.6

W. Metelmann, et al., RCMS, 14, 543 (2000)

Molecular ion region in the UVMolecular ion region in the UV--MALDIMALDI--TOF MS of TOF MS of peracetylated peracetylated NN--palmitoyl galactosyl sphingenene, palmitoyl galactosyl sphingenene, [M+Na][M+Na]+ + m/z m/z 932.6932.6

M = neutral molecular mass, M’ = M-HOAcC. E. Costello, unpublished data

32

MALDIMALDI--PSD mass PSD mass spectrum of spectrum of peracetylated peracetylated NN--palmitoyl palmitoyl galactosyl galactosyl sphingenene, sphingenene, [M+Na][M+Na]+ + m/z m/z 932.6932.6

M = neutral molecular mass, M’ = M-HOAc

C. E. Costello and J. E. Vathunpublished data

[M-2Neu5NAc+Na]+

LH

-Hex-HexNAc

LH[M(Na)-Neu5NAc+Na]+

1305.81

1277.78

1618.87912 65

940.681143.76

GD1a

Gal-(β1-3)-GalNAc-(β1-4)-Gal-(β1-4)-Glc-Cer

Neu5NAcα2-3Neu5NAcα2-3

750 1000 1250 1500 1750 2000 2250 2500

[M-2Neu5NAc+Cs]+

[M-2Neu5NAc+Na]+ LH[M(Cs)-Neu5NAc+Cs]+

[M(2Cs)+Cs]+

[M(2Cs)+Na]+

912.65

1415.73

1387.691838.72

2267.71

2151 79

m/z

LH

750 1000 1250 1500 1750 2000 2250 2500

2151.79

Low pressure positive-ion MALDI-FTMS spectra of GD1a and Na, Cs

33

Vibrational Cooling Vibrational Cooling MALDIMALDI--FTMSFTMS

Accumulation HexapoleAccumulation HexapoleGas Channels

h~ 3 Torr

at the spot

Ganglioside Ganglioside AnalysisAnalysis

GT1b1305.80

Gal-(β1-3)-GalNAc-(β1-4)-Gal-(β1-4)-Glc-Cer

Neu5NAcα2-3

Neu5NAcα2-8

Neu5NAcα2-3

VC MALDI FTMS allows intact

1000 1500 2000 2500

1277.771552.90

2245.02

[M(3Na)+Na]+

-2Neu5NAc(Na)1618.88

~10-4 mbar

desorption.

m/z

LH

-Neu5NAc(Na)1931.96

-3Neu5NAc(Na)1305.80

~1 mbar

1000 1500 2000 2500

PB O’Connor and CE Costello, RCMS, 2001,15, 1862PB O’Connor et al., JASMS, 2002,13, 402

34

Gangliosides desorbedGangliosides desorbedwith IR laser (urea, neg. mode)with IR laser (urea, neg. mode)GM1LH

GD2

٭٭٭٭

from stainless steelfrom stainless steel

GD2

LH

GD1aLH

-NeuAc(Na)

1000 1500 2000 2500

GT1b

m/z

LH(+urea)

V. Ivleva et al., Anal. Chem. 2004. 76, 6484.

Bovine whole brain gangliosidesBovine whole brain gangliosides

GD1VC MALDI-FTMS~3 mbar Desorption pressure (calc'd)337 nm N2 Laser (~50 J/mm)

GM1

GD1+HexNAc

-CO2-CO2

337 nm N2 Laser ( 50 J/mm)positive ions

asialo GM1GT1

1000 1250 1500 1750 2000 2250 2500

Mass/Charge (m/z)

+DHB+DHB

+DHB

35

MS/MS of GMMS/MS of GM1 1 from WBG mixturefrom WBG mixtureVC MALDI-FTMS~3 mbar Desorption pressure 337 nm N2 Laser (~50 J/mm)Negative ions-HexHexNAc Negative ions

-Neu5NAc

-H2O-H2O

-CH2CO

HexHexNAc

-H2O

-CH2CO

500 750 1000 1250 1500 1750 2000Mass/Charge (m/z)

[M-H]--Hex-Hex

Challenges of MALDI MS from surfacesChallenges of MALDI MS from surfaces

Determination of sample positionsDetermination of sample positions Determination of sample positionsDetermination of sample positions Sample location within surfaceSample location within surface Inadequate mixing with matrixInadequate mixing with matrix Inefficient transfer to membraneInefficient transfer to membrane Uneven surfaceUneven surface Inadequate penetration of laser beamInadequate penetration of laser beam Inadequate penetration of laser beamInadequate penetration of laser beam Compromised instrument performanceCompromised instrument performance Metastable decompositionMetastable decomposition

36

[M1+Na]+

Desorption from polymer membranesDesorption from polymer membranes

MS/MS of Gb3Cer [M+Na]+

D ti f t l

200 400 600 800 1000

O

OHHex-O-Hex-O-Hex

Y2

Y1 Y0

R

(Y0/O)''

Y2

Y1

Y0C3B3

C2B2

C1B1

*

*isobaric with internal fragment

MS/MS of Gb3Cer [M+H]+

Desorption from steel Sodiated Ions = oligosaccharide cleavages

Desorption from NafionTMO

NHO

UO

RLC

BRFA

200 400 600 800 1000

Y2'

[M1+H-H2O]+

Y1'

Y0

'Y0''

Y0' - CH2O

(Y0/O)' - CH2O (Y0/O)'

U(24:0)

3 [ ]Desorption from Nafion Protonated Ions = lipid cleavages

M. E. McComb et al., ASMS 2002

TLC plate

Matrix solution

TLC plateExtraction

E t ti l t

TLC-MS

Filter paperMembraneTLC plate

p

Blotting

Extraction solventi-PrOH/MeOH/H2O 0.2% CaCl240 7 20

Iron

TLCblotting-MS

p

Membrane

Matrix solution

37

Y3

Y2/C4

Y1Y2

Y2/B4

GalNAc Gal Gal Glc Cer

B1 B2 B3OH|

Cer = O-CH2CHCHCH=CH(CH2)nCH3

||

NHCO(CH2)mCH3

Globotetraosyl ceramide, GbGlobotetraosyl ceramide, Gb44CerCer

* matrix ions

1 280 13 20 136 0 14 00 1440 1480

1334.7

1362.3

[M+Na]+

18:1/24:0[M+Na]+

16:1/24:0Na23.1

*

***

*

*

*

UV TLC/MALDI-TOF MSGbGb44CerCer

1 280 13 20 136 0 14 00 1440 1480

100 200 300 400 500 600 700 800 1000 1200 1400 1600m/z

1036.3

***

1333 3

[M+Na]+16:1/24:0

1360.4[M+Na]+

18:1/24:0

Na

*IR MALDI-TOF MSTLCmembrane

1333.3

1280 1320 1360 1400 1440 1480

100 200 300 400 500 600 700 800 1000 1200 1400m/z

Na23.0 *

** ** *

J. Guittard, X. L. Hronowski and C. E. Costello, Rapid Commun. Mass Spectrom. 1999, 13, 1838-1849.

38

215 ng108 pmol

430 ng215 pmol

860 ng430 pmol

1075 ng538 pmol

Immunostained TLCs of mouse kidney glycolipids reacted with the 7A antibody (reactive with the Lewisx

determinant)

60

70

80

ity

159.2

274.1

199.2137.3

38.7177.3

18

20

22

24

26

28

3032

1924.8

2067.3

2052.02036.4

2024.4

2008.8

1940.6

*

**

*

* matrix ions

20

30

40

50

Rel

ativ

eIn

t ens

313.8976.9

900.2

22.814

16

18

1860 1 900 1940 1980 2020 2060 2100 214 0

200 400 600 800 1000 1200 1600 2000 2400Mass (m/z)

TLCblotting MALDI-TOF mass spectrum (DE, linear) of track with 215 pmol mouse kidney glycolipids; 2,5-DHB matrix, PVDF P membrane

39

Coupling VC MALDI FTMS with TLCCoupling VC MALDI FTMS with TLC

TLC plate

Actively Shielded 7T Superconducting Electromagnet

MALDI Ion Source, XY-Stage

RF-only HexapoleIon Guide

RF-onlyAccumulationHexapole

ElectromagnetXY-Stage

MALDI-FTMS

Turbo-pump

Turbo-pump

Turbo-pump

MALDI target

PreliminaryVisualizing

EA

[M-H]-

VC MALDI FTMS of TLCVC MALDI FTMS of TLC--separated gangliosidesseparated gangliosides

GM2

٭

[LH-H]-

GQ1b[M-H]-

[LH-H]-[M(K)-H]-

A

B

GM1 GM2 GM3 GD1a GD1b GT1b GQ1b mix mix

D

C

B

[ ( ) ]

[M(K)+K]-

GD1a[M+Na]+

[LH+Na]+[M+K]+

GT1b[M+Na]+

[LH+Na]+[M+K]+

[M(Na)+K]+C

DGD1aRp = 53,974

[M(Na)+K]+

GM1[M(Na)+Na]+[LH+Na]+

[M+Na]+

30001000 1500 2000 2500m/z

E٭ ٭

V. Ivleva et al., Anal. Chem., 2004, 76, 6484.

1887 1889 1891 1893

Rp 53,974

m/z

40

120 picomoles

GM1

LH

Detection limit of TLCDetection limit of TLC--VC MALDIVC MALDI--FTMSFTMS

Amount of GM1 spotted on TLC prior the separation.

٭

120 picomoles

12 picomoles (x3)

1.2 picomole (x4)240 femtomole

500 1000 1500 2000

٭ [M-H]-

m/z

٭

٭٭ ٭

120 femtomoles (x60)

12 femtomoles (x200)

1400 1500 1600 1700m/z

٭ = matrix clusters

TLCTLC--HP MALDIHP MALDI--FTMSFTMSDHB, CsI, TLC-separated

Leishmania lipids

1445

1577

1605

m/z Resolution

1 38 348321577

1200 1400 1600 1800 2000 2200

1445

17092172

C. E. Costello, V. Ivleva, U. Sommer, D. McMahon-Pratt, P. B. O’Connor, Desorption 2002

1577.385 34832

1578.390 35066

1579.394 35941

1580.397 34675

1581.508 37050

15771578

1579 1581

1577 1578 1579 1580 1581 1582 1583 1584 1585 1586

15821580

41

MALDIMALDI--FTMS (high resolution, MSFTMS (high resolution, MSnn) ) directly from TLC platesdirectly from TLC plates

Gb3Cer glycolipid desorbed directly from TLC plate

RP 30,000

MS

MS2

MS3 -Hex

-Hex

972968

1355 1360 1365 1370

MS4

400 600 800 1000

-Hex

E. Mirgorodskaya et al., ASMS 2001

Combination of IRMPD & SORICombination of IRMPD & SORI--CIDCID

Gal—O—Gal—O—Glc—O—Cer

Y1 Y0Y2MS [M+Li]+1142.8

1135 1140 1145 1150

1140.8

1138.8Cer = O-CH2CHCHCH=CH(CH2)nCH3

|NHCO(CH2)mCH3

600 605 610 615

200 300 400 500 600 700 800 900 1000 1100 1200100 m/z

200 300 400 500 600 700 800 900 1000 1100 1200100 m/z

MS / MS(IRMPD)

[M+Li]+

Y0 (Cer)

Cer-48606.6

604.6

1135 1140 1145 1150

608.6

Y1

Y2

(Cer-48)'

LCB (18:1)258.3

FA (24:0)374.4

200 300 400 500 600 700 800 900 1000 1100 1200100 m/z

MS / MS(IRMPD) / MS(SORI-CID)

E. Mirgorodskaya et al., ASMS 2001

42

Summary of TLCSummary of TLC--VC MALDIVC MALDI

TLC-VC-MALDI-FTMS of glycolipids is simple, accurate.

External MALDI source avoids deleterious effects of surface irregularities.

IR & UV VC MALDI both minimize decomposition.

Broadband resolving power > 50,000; accuracy > 1.5 ppm

Detection limit is ~100 fmol deposited on plateDetection limit is ~100 fmol deposited on plate.

Tuning ion source pressure allowed isomer differentiation.

VC-MALDI QoTOF MS shows similar advantages for glycolipids.

Advantages of derivatization before Advantages of derivatization before MS analysis of glycoconjugatesMS analysis of glycoconjugates

Sensitivity increase Sensitivity increase

Mass shifts characteristic of functional groupsMass shifts characteristic of functional groups

Control over fragmentation pathwaysControl over fragmentation pathways

Location of f nctional gro p sitesLocation of f nctional gro p sites Location of functional group sitesLocation of functional group sites

Conversion of oligosaccharides to glycolipidsConversion of oligosaccharides to glycolipids

43

Summary: MS approaches for lipids and Summary: MS approaches for lipids and glycoconjugates glycoconjugates an increasing range of an increasing range of options options Lipidomics, Glycomics!Lipidomics, Glycomics!

DerivatizationDerivatization OnOn-- and offline separationsand offline separations

––

ESIESI--QqQ MS and MS/MS, ESIQqQ MS and MS/MS, ESI--QIT MSQIT MSnn

MALDIMALDI TOF MS and PSDTOF MS and PSD MALDIMALDI--TOF MS and PSDTOF MS and PSD ESI and MALDI QoTOF MS and MS/MSESI and MALDI QoTOF MS and MS/MS ESI and VC MALDI FTESI and VC MALDI FT--ICR MSICR MSnn

ESI LTQESI LTQ--OrbitrapOrbitrap

References References

A. L. Dill et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Dec 31. [Epub] E. Fahy et al., J. Lipid Res., 46, 839.. (2005) X. Han and R. W. Gross, J. Lipid Res., 44, 1071 (2003)

P M H t hi t l J Li id R 49 804 (2008) P. M. Hutchins et al., J. Lipid Res., 49, 804 (2008) V. Ivleva et al., Anal. Chem., 76, 6484 (2004) R. A. Johanson et al., Anal. Biochem. 362, 155 (2007) W. Metelmann et al., Rapid Commun. Mass Spectrom., 14, 543 (2000) S. Milne et al., Methods, 39, 92 (2007) T. W. Mitchell et al,, J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jan 21. [Epub] R. C. Murphy, Chem. Rev. 101, 479 (2001) P. B. O’Connor and C. E. Costello, J. Am. Soc. Mass Spectrom., 13, 402 (2002) M Pulfer and R C Murphy Mass Spectrom Rev 22 332 (2003) M. Pulfer and R.C. Murphy, Mass Spectrom. Rev., 22, 332 (2003) U. Sommer et al., J. Lipid Res., 47, 804 (2006) M. C. Thomas, TW. Mitchell, DG Harman, JM Deeley, JR Nealon, SJ Blanksby, Anal.

Chem., 80, 303 (2008) P. Whelan et al., J. Am. Soc. Mass Spectrom., 7, 140 (1996)

mass spectrometry of lipids and glycoconjugatesj chromatogr b analyt technol biomed life sci. 2009,...

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