Mass Spectrometry: A Flexible Tool for Drug DiscoveryTool for

Recombinant Protein / Peptide Characterization––– In-gel digestion + nano-LC/MS/MS– Quantitation – Amino Acid Analysis (LC based methodology)– Phosphorylation site mapping–

Cellular lysates– Chemoproteomics

Tissues / Cell Media / Plasma– Quantitative LC/MS/MS of endogenous peptides and smallQuantitative LC/MS/MS of endogenous peptides and small

molecules

Protein LC/MS Open-Access

Non-covalent MS

Access

1D SDS Page Gel

– Multiplexed technique every– Multiplexed technique, every biochemists knows how, slow (1 hr per run), mass accuracy ~1%

MALDI-TOF100

041404_001 6 (1.074) Sm (SG, 2x10.00); Cm (6) TOF LD+ 39332598.441

32947.305MALDI-TOF

Fast (minutes), unlimited mass range, easy to use, requires some sample preparation mass accuracy 0 1

%

32250.41233285.121

preparation, mass accuracy 0.1 –0.01%, mass resolution ~500

ESI-MS (ESI-TOF) 100

5332 67 (3.619) M1 [Ev-77914,It10] (Gs,0.750,450:2200,0.75,L33,R33); Cm (63:72) 1: TOF MS ES+ 1.16e332601.0000

32940.0000

30000 30500 31000 31500 32000 32500 33000 33500 34000 34500 35000 35500m/z0

32005.70730173.545

33538.016

33976.930

34414.379 35569.594

– Excellent mass accuracy and mass resolution (<0.01% and > 1500), requires desalting, limited mass

/% 32260.5000

33278.2500

range, m/z spectra need to be deconvoluted

30000 30500 31000 31500 32000 32500 33000 33500 34000 34500 35000 35500mass0

31437.000030926.2500

33618.7500

35288.250034576.5000

Enable sample submitters access to instrumentation with minimal user training and analyst involvementUser friendly system Robust LC/MS analysis method Instrument start-up and shutdown between analysesp yWeb-based sample submissionSample registration completed at appropriate instrument Automated processing using a dedicated serverAutomated processing using a dedicated serverIntelligent data processing and results reviewResults automatically posted to website Works with multiple vendors

The Black Box

Auto-Interpreted Report back to the biologistAuto-Interpreted Report back to the biologist

Theoretical MW = 29024.4

Sample RegistryInstrument Computer

TOF Data Acquisition

OA LC/MS login

q

Data File

Processing Server

Open Access Monitor

Integration, m/z spectra

Moved Data File

Deconvolution Report

Deconvolution

Auto Interpretation

QC of fractions and purified recombinant proteins– Determine if N-term is modified or lost based on differences– Determine if N-term is modified or lost based on differences

between expected and experimental MW– Look at extent of expression modification

Incorporation of covalent modifications (kinetics or end point)Incorporation of covalent modifications (kinetics or end-point)– Phosphate– Biotin – Ligands

Analysis of Products from limited proteolysis– Typically can deduce fragment sequence based on mass and yp y g q

enzyme specificity

Theoretical MW = 274,631.3(difference due to an unspecified phosphorylation site)phosphorylation site)

R. Klein, UNC Ph.D. Thesis

R Klein UNC Ph D ThesisR. Klein, UNC Ph.D. Thesis

Inhibition of ErbB family receptor tyrosine kinases (EGFR, ErbB-2) represents a major advance in the

NH Cl

OF

Hrepresents a major advance in the treatment of solid tumors,

– Reversible ATP competitive EGFR (gefinitib and erotinib)Reversible ATP competitive

O N

N

NH

MeO2S

lapatinib F

– Reversible ATP competitive EGFR and ErbB-2 – lapatinib (Tykerb)

– Irreversible binder at Cys797 in EGFR Cys 805 (ErbB-2) and

N

NH

NNH

OO

Cl

EGFR, Cys 805 (ErbB 2) and Cys803 (ErbB-4) - canertinib and pelitinib

Identify efficacious EGFR/ErbB-2 inhibitors that are structurally distinct

N

O

canertinib(CI-1033)

inhibitors that are structurally distinct relative to lapatinib. Several anologs of thieno[3,2-d]pyrimidines and thieno[2,3-d]pyrimidines were found to be potent inhibitors of purified EGFR d E bB 2 d th

RNS

NH

O

Cl

F

N

NH

O

Cl

F

REGFR and ErbB-2 and the proliferation of tumor cells that highly express these kinases.

Wood, E.R. et al. PNAS (2008) 105, 2773

N NSR

[3,2-d] Thienopyrimidines [2,3-d] Thienopyrimidines

Wood, E.R. et al. PNAS (2008) 105, 2773

100

120

40573 (EGFR (678-1020)

SO O

NH

O

N

N

NH Cl

OF

GFR

Mod

ified

(% T

otal

)

20

40

60

80

41058

N

N

NH

ON

O

Canertinib MW = 486 Da

Time (minutes)

0 50 100 150 200 250 300 350

E

0

20

C d S i R % Alk l i fNH

Cl

O

F

41050

Shift = 485 Da Cmpd Series R % Alkylation ofEGFR

3 h 20 h6 A H 9 67

7 A 0 15

N

NS

NH

O

Cl

F

NH

Thienopyrimdine MW = 478 DaShift = 477 Da

NH

NH2

N

NN

H

H

7 A 0 15

8 A 83 100

9 A 0 0

40000 40400 40800 41200 41600mass

Wood E R et al

NH2

NH

NH

11a B 0 19

12 B 4 24

13 B 0 0O OWood, E.R. et al.

PNAS (2008) 105, 2773

NN

H

4 - - 100 100RN

NS

NH

O

Cl

F

N

N

S

NH

O

Cl

F

R

[3,2-d] Thienopyrimidines [2,3-d] Thienopyrimidines

NN

S

N N

N

S

NHAr

H

HHS

NS

NHAr

N

H

HH+

N

N

SH

H

S NHAr

NH

Wood, E.R. et al. PNAS (2008) 105, 2773

NH2-FGC-( )-OH, m/z 402.62+

W d E R t l PNASWood, E.R. et al. PNAS (2008) 105, 2773

Screened over 800 compounds at two time points (3 and 20h)

The [3,2-d] thienopyrimidines had higher reactivity than the [2,3-d] series

Enabled program to select lead compounds for additional testing

Binding experiments were conducted before and after installation of OA software

–

–

Confirmed site of covalent bond attachment to EGFR

Other program teams have utilized Open-Access MS and LC/MS/MS sequencing to determine the presence and location of covalently boundsequencing to determine the presence and location of covalently bound compounds – prior to doing structural studies

Protein Ligand+ Protein expressed with ligand or native ligand

Ligand+

ligand or native ligand

Incubation time

Vary incubationincubation time &/or solvent

conditions

Veramyst/Advair/Flonase

Avandia

SF-1 and LRH-1 native ligand studies

SF-1 and LRH-1 are in NR sub family 5AActive in many cell typesActive in many cell typesNot regulated by steroid, retinoid, other non-polar ligands“Obvious ligand candidates have not been forthcoming”

I. Krylova et al. Cell (2005) 120, 343-355

What is the identity and quantity of the native ligands found in bacterially expressed systems of mouse and human SF-1 and LRH-1?

Can the native ligand be removed and exchanged with a potential native ligand found in eukaryotic cells?

Separate free from bound ligands using gel filtration “spin columns” and analyze bound fractions using reversed-phase LC/MSand analyze bound fractions using reversed phase LC/MS

– Detect ligand only – Small molecules of interest may not ionize

Directly monitor intact protein-ligand complex using electrospray mass spectrometry

f– Membrane filtration– Size-exclusion

Spin columnsIn-line gel columns (Cavanagh et al. Anal. Chem 2003)

– Automated and optimized using standard LC equipment (G.M. Waitt, et al. JASMS (2008) 19, 239)

Protein

Protein

Protein

Protein

SaltSalt

Salt

SaltSalt

Salt

ProteinProtein

BioRad P6DG Sephedex G25 BioRad P6DG0.75mm ID

p1.6mm ID 1.6mm ID

G.M. Waitt, et al. JASMS (2008) 19, 239

Cap LCChannels A & BWaste W t

V

P6 G l C l

Channels A & B

AutosamplerNanoLC

Waste Waste

P6 Gel ColumnLC

ESI

Mass Spectrometer

ESI

Mobile Phase 20 mM NH4OAcCh l A+B Fl R t 10 L/ i Mass SpectrometerChannels A+B Flow Rate 10 L/min. Nano LC Flow Rate 4 L/min.Valve Switch ~ 8 min. Run-time 20 min.


4 .0 E + 0 5

3 .0 E + 0 5

3 .5 E + 0 5

2 .0 E + 0 5

2 .5 E + 0 5

1 .0 E + 0 5

1 .5 E + 0 5

5 .0 E + 0 40 5 0 1 0 0 1 5 0 2 0 0

10 mM ammonium acetate ( ), 10 mM Tris HCl ( ), and 20 mM Tris, 200 mM NaCl at pH 8 ( )


2796328711

100

[A][B] 2872

2797

[B]10+

[A]10+

9

3191

3108

[B]9+

[A]9+

%

28684

25432611

3108

28963217

[A]11+[B]11+

% 2611 2947 3217

m/z

27980 28725

27600 28000 28400 28800 29200 296000mass


745.5007745.5007

719 4851714.5060 719 4851714.5060 719.4851714.5060

688 4898

%

719.4851714.5060

688 4898

%

773.5309759.5165

733.5007

688.4898

773.5309759.5165

733.5007

688.4898

690 700 710 720 730 740 750 760 770 7800

702.5049

690 700 710 720 730 740 750 760 770 7800

702.5049

690 700 710 720 730 740 750 760 770 780m/z

690 700 710 720 730 740 750 760 770 780m/z

281 02000 281 02000253.0

281.0

1500

2000253.0

281.0

1500

2000

OHPO

OOH OH

417 11000

Intensity.

417 11000

Intensity.

PO

O

C18H33O2

OP

O

OO

OHO

PO

OO

OH

417.1

491.1

509 2

745.5500

417.1

491.1

509 2

745.5500C18H33O2

C18H33O2C16H29O2

389.0 463.1

509.2

0300 400 500 600 700

m/z

389.0 463.1

509.2

0300 400 500 600 700

m/z

745.5007 745.5025 C40H74O10P- C16:1, C18:1 PG719.4868 719.4851 C38H72O10P- C14:0, C18:1

C16:0, C16:1PG

714 5060 714 5074 CH NO P- C16:1 C18:1 PE714.5060 714.5074 CH73NO8P C16:1, C18:1 PE691.4562 691.4550 C36H68O10P- C12:0, C18:1

C14:0, C16:1C14:1, C16:0

PG

,773.5309 773.5333 C42H78O10P- C18:1, C18:1 PG759.5165 759.5176 C41H76O10P- C18:1, C17:1

C16:1, C19:1PG

747 5136 747 5181 C H O P- ND3 PG747.5136 747.5181 C40H76O10P ND3 PG688.4898 688.4852 C37H71NO8P- ND PE733.5007 733.5025 C39H74O10P- ND PG702.5049 702.5009 C38H73NO8P- ND PE0 50 9 0 5009 C38 73 O8742.5359 742.5392 C41H77NO8P- ND PE

System Protein Ligand H6-H7Conc. (μM) Conc. (μM) SequenceConc. (μM) Conc. (μM) Sequence

mSF-1 30 34 VQAGSLLHShSF-1 50 23 TQAGSLLHSmLRH-1 30 4 SHTEVAFNNhLRH-1 30 28 SQAGATLNNmSF-1 A270W* 30 0.3 VQAGSLLHS

*M i l d i H3*Mutation located in H3


100

9703_002 31 (5.276) M1 [Ev-27332,It8] (Gs,0.750,2469:3286,0.75,L33,R33); Cm (30:35) TOF MS ES+ 1.31e330562.7500

30739.7500

%

31220.500031044.250031252.7500 PG ligands absent

30600 30800 31000 31200 31400 31600mass0

No binding


Utilized various mass spectrometric techniques to identify, quantitate, and monitor the exchange of native ligands from SF-1 and LRH-1and monitor the exchange of native ligands from SF 1 and LRH 1

With these and other biochemical data, binding activity for mLRH-1 was uniquely diminished and alteredwas uniquely diminished and altered.

Proposed that phospholipids regulate gene expression by directly binding to NR5A nuclear receptorsbinding to NR5A nuclear receptors.

L. Yin, et al., Science (2007) 318, 1786-1789

Mass reconstruction of +TOF MS: 0.101 to 0.801 min from 20509_001.wiff Max. 129.7 cps.

39473.8086

+TOF MS: 2.519 to 2.607 min from 20509_001.wiff Agilent Max. 1.2e5 counts.

8.4e4

39473.8

616.17

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4616.17

615 1 Da39486.1289

39497.128938858 6914

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

38858.7

615.1 Da

38858.6914

614 5 615 0 615 5 616 0 616 5 617 0 617 5 618 0 618 5 619 0 619 5 620 00.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

614.5 615.0 615.5 616.0 616.5 617.0 617.5 618.0 618.5 619.0 619.5 620.0m/z, amu

Mass reconstruction of +TOF MS: 0.301 to 0.751 min from Sample 2 (20512) of 20512_001.wiff Max. 33.6 cps.

39482.2813


1.6e4

1.7e4

1.8e4

1.9e4

2.0e4 616.1739428.3

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

617.6 Da

39509.1211

37801 9219 612.0 613.0 614.0 615.0 616.0 617.0 618.0 619.0 620.0 621.0 622.0 623.0 624.00.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

37801.937801.9219

39550.8086 m/z, amu


1 3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5 620.21

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

618.0 619.0 620.0 621.0 622.0 623.0 624.0 625.0m/z, amu

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4


Mass reconstruction of +TOF MS: 0.251 to 1.051 min from 20510_001.wiff Max. 36.7 cps.

39788.4102

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

39788.4

616.17

39835.9297

39879.2695

39706.2383

39919.7500 6000 0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

632 8 Da?

39962.761739665.8711

613.0 614.0 615.0 616.0 617.0 618.0 619.0 620.0 621.0 622.0m/z, amu

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0


632.8 Da?

40003.2383

37155.5586

37219.9102

_ g

8000.00

8500.00

9000.00

9500.00

1.00e4

1.05e4

1.10e4

1.15e4

37155.6 629.17

3500 00

4000.00

4500.00

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

Inte

nsity

, cou

nts

625.0 626.0 627.0 628.0 629.0 630.0 631.0 632.0 633.0 634.0 635.0 636.0 637.0 638.0m/z, amu

0.00

500.00

1000.00

1500.00

2000.00

2500.00

3000.00

3500.00

siRNA and other experiments demonstrated that Rev-Erbappears to play a role in basal suppression of gluconeogenic genes

Even for WT Rev-Erb , hemin was i d f it t f NC Rrequired for recruitment of NCoR

and HDAC3


Gluconeogenic genes and glucose production in liver cells are affected by the presence of heminby the presence of hemin

RevErb appears to function as a receptor for hemeThis study suggests that it may play a central role in coordinating

t b limetabolic processesOther work suggests RevErb plays a role in circadian rhythms– Clock proteins NPAS2 and PER2 have Heme as a prosthetic

group as wellgroup as wellMay make an interesting drug target for metabolic diseases


Exploring high throughput mass spectrometry technologies to support assay development and MOA studiesassay development and MOA studies

– BioTrove RapidFire– ABI FlashQuant

Prosolia DESI (Desorption ElectroSpray Ionization)– Prosolia DESI (Desorption ElectroSpray Ionization)ETD (Electron Transfer Dissociation)

– Peptide sequencing – Phosphomapping– Intact protein sequencing

Interesting “one-off” analyses g y

Erin ChaneyJohn Hall

UCSF– Holly Ingrham

Mary MoyerGreg WaittCraig WagnerW d Whit

– Irina Krylova

Penn Mit h LWendy White

Scott DickersonKevin Madauss

– Mitch Lazar– Lei Yin

Kevin MadaussLisa ShewchukShawn WilliamsRobert Xu

Ken PearceBruce WiselyEddi W dEddie Wood

David Uehling

For Research Use Only. Not for use in diagnostic procedures.

Mass Spectrometry: A Flexible Tool for Drug DiscoveryTool for

Documents

Transcript of Mass Spectrometry: A Flexible Tool for Drug DiscoveryTool for