Applications of mass spectrometry to cardiovascular research

Barnes-January 21, 2003

Applications of massspectrometry to

cardiovascular researchStephen Barnes, PhD

Department of Pharmacology &Toxicology, UAB


Overview of the talk

• Vaporizing peptides, proteins and heatsensitive compounds

• Separating and identifying proteins

• Confirmation of protein identity andposttranslational modifications


Congratulations to theCongratulations to theNobel Laureates - 2002Nobel Laureates - 2002

"for the development of methods for identification and structureanalyses of biological macromolecules"

and"for their development of soft desorption ionisation methods formass spectrometric analyses of biological macromolecules"

John Fenn Koichi Tanaka


Electrospray Electrospray Ionization (ESI)Ionization (ESI)

VacuumAtmospheric pressure

MassAnalyzer

N2 curtain gas

+HV

nebulizinggas

sample solution

++ +

+ +

+ +

++

+++

++

++

[M + nH]n+

++

1. Solvent evaporation2. Coulombic repulsion


Use of LC-MS to measure Use of LC-MS to measure isoflavonesisoflavones

241/119

253/223

269/133

0 1 2 3 4 5 6

Time (min)

100

50

0

Rel.

Int.

(%) 7260

100

50

0

Rel.

Int.

(%) 8625

100

50

0

Rel.

Int.

(%) 9745

Standards

Equol

Daidzein

Genistein

0 1 2 3 4 5

Time (min)

100

50

0

Rel.

Int.

(%) 238

100

50

0

Rel.

Int.

(%) 23,590

100

50

0

Rel.

Int.

(%) 16,860

241/119

253/223

269/133

Equol

Daidzein

Genistein

Rat serum


LC-MS analysis of LC-MS analysis of prostanoidsprostanoids

351/271

335/195

295/277

319/115

0.0 2.0 4.0 6.0 8.0 10.0

Time (min)

100

50

0Rel

. In

t. (

%) 525

100

50

0Rel

. In

t. (

%) 605

100

50

0Rel

. In

t. (

%) 430

100

50

0Rel

. In

t. (

%) 780

PGE2

Leukotriene B4

13S-HODE

5S-HETE


NanoElectrosprayNanoElectrospray

5 mm


LC-MS of peptide mixtures

Q1 Q2

Collision gas

Electrostaticreflector

TOF detector

Analytical reverse phase column75 mm i.d. x 15 cm

Flow rate 200 nl/min

Acetonitrile gradient

pre-columnfor desalting

waste

Loadsample


• Hydrolyze everything!

• For a cell expressing 5,000proteins, this leads to>100,000 peptides

• Can be fractionated, but still10,000-20,000 to differentiate

• Enormous bioinformaticsproblem

MUDPIT - MUDPIT - MUMUltilti--DDimensionalimensionalPProtein rotein IIdentification dentification TTechnologyechnology

MS-MS analysison Qqtof

Massive computing

nanoLC

0-40% MeCNgradient

10 mM

20 mM

40 mM

60 mM

80 mM

100 mM

NaCl (1-200 mM step gradient)

200 mM

Cation exchangecolumn (H+)

John Yates


Matrix-Assisted LaserDesorption Ionization (MALDI)

Flight tube and drift region tomeasure the time-of-flight (TOF)

Accelerating pulse

Short laserpulse

detector


Posttranslational modifications

Differential mRNAsplicing accountsfor a small part ofthe protein formsarising from a gene- the remainder aredue to chemicalalterations of theprotein AFTER it’sbeen translated

- leader sequence

farnesylation

N-acetylation

phosphorylation

oxidation


Posttranslational modifications

• some are permanent (removal of N-terminal Met, N-glycosylation atasparagine, farnesylation on cysteines)

• some are transient (phosphorylation, O-glycosylation on Ser, Thr and Tyr)

• others are unintended (nitration,oxidation)


How can we detect PTMs?

Combination of:– isoelectric focusing (pI changes of

single proteins) and electrophoresis

– affinity isolation (to detect all proteinswith a specific chemical grouping)

– mass spectrometry (molecular weightchanges)


Separating proteins in twodimensions

IEF

SDS-PAGE


Detecting a modification using 2DE

• Adding a phosphate residue to a serine,threonine or tyrosine residue increases theacidity of the protein, i.e., its isoelectric point (pI)decreases with only a small change in MW– This causes the protein to shift horizontally in 2DE

• Adding a ubiquitin residue to a specific lysineresidue adds over 8 kDa to the MW– This causes the protein to be shifted upward (higher

MW)


Differential protein labeling with Cy3 and Cy5Superimposed images from the same gel

of normal and cancer cell lines from the breast

Courtesy of Helen Kimand Jessy Deshaine


WeWe’’ve identified a spot -ve identified a spot -what next?what next?


WaterBath37oC

Speed-Vac

trypsinIncubate

overnight 1:20

Peptide finger print mapping

destain

Eppendorftube

MALDI plate

DesaltingZiptip

684.

1178

9.57

1048

.72

1122

.79

1165

.80

1277

.83

1461

.00

1705

.16

1838

.26

1937

.22

1994

.24 21

37.2

122

84.2

523

84.2

7

2647

.50

00

5000

10000

15000

Co

un

ts

1000 1500 2000 2500 3000

Mass (m/z)

MALDI-TOFanalysis


Peptide fingerprint mapping

• Assuming a 1 Da mass measurement accuracy,“theoretically” it takes on average 2.1-2.3 peptidemasses to uniquely identify a protein from adatabase of all possible peptides (generated insilico) resulting from action of a specific peptidase– In practice, 4-6 peptides are generally needed

• Analysis of a 2DE protein spot requires 100 fmol (5ng of a 50 kDa protein)– Smaller amounts can be detected, but coverage is low


D masses from modifications

Change (Da) Chemical type

-79 5' dephospho

-58 Desmosine (from Lysine)

-48 decomposed carboxymethylated Methionine

-44 decarboxylation of gamma carboxy Glutamate

-43 gamma-glutamyl semialdehyde (from arginine)

-42 Ornithine (from Arginine)

-34 Lysinoalanine (from Cysteine)

-34 Lanthionine (from Cysteine)

-34 Dehydroalanine (from Cysteine)

-30 Homoserine formed from Met by CNBr treatment


D masses from modifications

D Da Chemical change

78 3-Bromination (of Tyrosine with 79Br)

78 L-O-bromination of Phe with 79Br

80 L-O-bromination of Phe with 81Br

80 Sulphonation (SO3H) (of PMC group)

80 Sulphation (of O of Tyrosine)

80 Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N-epsilon of Lysine)

80 3-Bromination (of Tyrosine with 81Br)

For all the others, http://www.abrf.org/index.cfm/dm.home


Tyrosine modifications

tyrosineCH2

OH

CH2

OH

CH2

OH

CH2

OHCH2

OH

CH2

OHCH2

OHCH2

OH

NO2

Cl

Br

3’-nitrotyrosine

3’-chlorotyrosine

3’-bromotyrosine


Site-specific nitration of a tyrosine-Site-specific nitration of a tyrosine-containing peptide using CID MS-MS spectracontaining peptide using CID MS-MS spectra

100

75

50

25

0

Rel.

Int.

(%)

100 150 200 250 300 350 400 450 500 550 600 650

m/z

36

27

18

9

0

Rel.

Int (

%)

129

147 183

200

240

258

276

291

408

448

484

537594

611 647

682

129147

183

200

240

258276 363

403

439

492 549567

637

y5

y4b4

y3b3

b2

y2y1

200 408 537 665

NO2

A Q Y E K683 612 484 276 147

200 363 492 620

A Q Y E K638 567 439 276 147

b2

y1

y2 b3

y3

b4y4

y5

+45

nitrated protein

control

Crow et al (1997)


AcknowledgmentsAcknowledgments

• Jessy Deshaine

• Kenneth Jones

• Helen Kim, PhD

• Marion Kirk

• Heath McCorkle

• Ray Moore

• Landon Wilson

Support from:

• NIH to Purdue-UABBotanicals Center forAge-Related Research

• NCI to UABComprehensiveCancer Center

• NCRR for SharedInstrument Awards

Applications of mass spectrometry to cardiovascular research

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