Proteomic Assessment of Thiol Modifications

Victor Darley-Usmar, Ph.D.Center for Free Radical Biology, University of Alabama at

Birmingham

Increased protein modification in

cell signaling or oxidative stress

ROS/RNS

Modified proteins (altered function)

nitrotyrosinethiol modification

carbonyl formation

Proteomics is the study of a protein complement in response to a stimulus

Potential for biomarkers Defining mechanisms

Hypothesis Generation

Some Reactive Proteomes In Free Radical Biology

Thiol

Nitro Carbonyl

Electrophile

Role of thiols in protein function and cell signaling

“redox signaling”

Thioredoxin catalyzes the S-nitrosation of the caspase-3 active site cysteine.Mitchell DA, Marletta MANat Chem Biol. 2005 Aug;1(3):154-8. Epub 2005 Jul 10.

–S-

–S-

RSH

–SH

–S–S–R

–S

–S

–SNO

–SH

NO, RNS

–S–S–

–SH

HS–

=O

–SH

ONOO-

–SNO ROS

ROS

=O

–S–OH

=O

–SOH

–SH

–SH

=O

–S–OH

–SH

Cooper et al. Trends Biochem. Sci. 2002

–SH

–SR

H

O

OH

Sub-Classes of the Thiol Proteome

–S-

–S-

RSH

–SH

–S–S–R

–SNO

–SH

NO, RNS

–S–S–

–SH

HS–

ROS

–SOH

–SH–SH

–SR

Modifications Discussed

O

–SX

–SX

–S- tag –S-tag

signal

Western blot/

Imaging

–SX

ROS/RNS

signal

Step 1: Are thiols modified at all?

Biotin as a tag

N-(biotinoyl)-N'- (iodoacetyl)-

ethylenediamine(BIAM)

Advantages

Wide range of commercially synthesized tags available.

extremely sensitive when coupled with streptavidin/HRP

Can be used to pull down targets

Can be quantitative

Less sensitive to local protein environment (c.f. antibodies)

Biotin as a tag

N-(biotinoyl)-N'- (iodoacetyl)-

ethylenediamine(BIAM)

Disadvantage:Endogenous carboxylases

105K

75K

b

t-15

d-P

GJ 2

Biochem J 394:185-95 (2006) MitochondriaB

IAM

Cells

BIA

M

Cytochrome c: small (12,000 Da), water soluble, multiple surface lysine residues.

Cytochrome c as an internal standard for protein and Biotin

Biotin Tagging through Lysine:

Native Cytochrome c - 12360

Matrix Adduct - 12569

10000.0 12000 14000 16000 18000 20000Mass (m/z)

3 Biotins - 13374

4 Biotins - 13713

5 Biotins - 14052

2 Biotins - 13034

6 Biotins - 14391

7 Biotins - 14731 Apomyoglobin Standard169528 Biotins - 150681 Biotin, 1 K -

12733

bt cyt.c

17588189Biotin (pmol)

0 20 40 60 80 1001201401601800

500

1000

1500

2000

2500

3000

Ban

d D

en

sit

y (

Arb

itra

ry U

nit

s)

Biotin (pmol)

Free Radic Biol Med. 40(3):459-68 (2006)

N-(biotinoyl)-N'- (iodoacetyl)-ethylenediamine

(BIAM)

Anal. Biochem. 283:214-221, 2000

Step 1:Prepare the sample and analyze by 1D-SDS-PAGE

detect biotin (Western)

Treatment

lyse sample with BIAM at pH 8.0-8.5

Biochem J 379:359-366, 2004

Sypro Ruby stain biotin blot

Step 2: Application to a 2D-Proteomic Format

(Rat Liver Mitochondria)

AbundanceProtein amt x dye binding

Thiol ProteomeProtein Amt x SH groups x reactivity

Biotin tag is more sensitive than the Sypro Stain

-bt-bt

bt-

protein biotin

0.3μg 0.01μg

abundance proteome is not the same as thiol proteome

S-Bt

S-BttB-S

S-B

t

S-Bt

S-B

t

–S-

–S-

RSH

–SH

–S–S–R

–SNO

–SH

NO, RNS

–S–S–

–SH

HS–

ROS

–SOH

–SH–SH

–SR

O

–SX

–SX

Diagonal electrophoresis for inter-protein disulfides

hi

low

SH

SH

S S

oxidativestress

exciselane

Identify proteins off of diagonal

N-terminal Edman degradation sequencing

Mass spectrometry

Immunoblot and probe for candidate proteins

S S

S

S

Reduce

S

S

Adapted from J Biol Chem. 2004 Oct 1;279(40):41352-60.

–S-

–S-

GSHCys

–SH

–S–S–R

–SNO

–SH

NO, RNS

–S–S–

–SH

HS–

ROS

–SOH

–SH–SH

–SR

O

–SX

–SX

-biotin protein S- HS-X

protein S-

oxidizingenvironment

-biotinS-X

detection, purification, imaging, identification using

avidin-based methodologies

GSHGSH esterCys

X =

Protein Biotin

–S-

–S-

RSH

–SH

–S–S–R

–SNO

–SH

NO, RNS

–S–S–

–SH

HS–

ROS

–SOH

–SH–SH

–SR

O

–SX

–SX

Differences in structure due to PTM of SH group in Biology are subtle

Surrounding amino residues will lead to epitope bias

S

N O

RSOH

RSO2H

S-nitrosothiol Sulfenic Sulfinic

StrategiesDirect detection of the PTM.

Antibody: epitope too small and not structurally distinct. Mass Spectrometry: Sensitivity often not adequate

Differential chemical properties leading to specific insertion of a tag.

protein

SNO

SOH-S Sulfenic acid

S-nitrosothiol

StrategiesDirect detection of the PTM.

protein SOH-S

proteinSOH

protein

Does not react with thiol, sulfinic, sulfonic, disulfide,GSNO, Met Sulfoxide groups.

Sulfenic Acid

Dimedone

Strategies

Differential chemical properties leading to specificinsertion of a tag.

protein

SNO

SOH-S

BIOTIN SWITCH

protein

SNO

SOH-S

Alkylation to block free S-

Remove alkylatingagent

protein

SNO

SOHRS

ascorbate reduction

protein SNO protein SOHR-S R-S

arsenite reduction

Remove reagents

Restore the SOH or SNO to S-

protein SBT protein S-BTR-S R-S

TAG

AFFINITY PURIFY and DETECT

Examples of RSNO/RSOH

RSNO in endotoxin trtdmacrophage

Biotin Protein

RSOH in peroxide (100 M)treated heart

Biotin

pHProtein stained gel

detect biotin Reactive Thiols

Lyse and treat cells (BAEC)

with BIAM

DetaNONOate

2D-IEF

How abundant are S-nitrosated Proteins?

150

10075

50

35

30

15

10

3 10pH

3 10pH

Control-SH Blot After NO treatment-SH Blot

Master map

Total spots = 135Matched =41

Matched

Unmatched

PNAS. 2004:101(1):384-9

70% thiolsmodified

Measure RSNO and thiols by direct non-proteomics

technique.

RSNO 11.2 ± 0.07pmol/mg protein

Protein Thiol approx 40-80 nmol/mg protein

0.014-0.028%

The problem of false positives

S- SR SX

30% SX PTM in a population of 20 proteins

Convert Tag

STag

False Positiveis 14%

Block 93%effic.

The problem of false positives

S- SR SX

5% SX PTM in a population of 20 proteins

Convert Tag

STag

False Positiveis 50%.

Block 93%effic.

–S-

–S-

RSH

–SH

–S–S–R

–SNO

–SH

NO, RNS

–S–S–

–SH

HS–

ROS

–SOH

–SH–SH

–SR

Detecting Specific Modifications

O

–SX

–SX

ADPATP

H+

H+e- O2

H+

H+

H+H+

H+

H+H+

H+H+ H+

Future Directions; organelle specific

P I+

IBTP+

–SH

–S–TPP

IgG

Control EthanolAnti-IBTP

Aldehyde dehydrogenase

HSP70

2D SDS-PAGE followed by western blotting

1 Pyruvate carboxylase 129.6 195 28/492 Hsp70 72.1 194 28/743 Hsp60 60.9 90 8/134 Glutamate dehydrogenase 56 98 8/155 Protein disulfide isomerase 56.9 123 10/96 Mitochondrial aldehyde dehydrogenase 53 135 12/157 Acetyl-coenyzme A acyl transferase 2 41.8 79 7/13

Mass (kDA)

MOWSE score

No. peptides matched/unmatched

12

34

56 7

Am J Physiol Gastrointest Liver Physiol. 2004 Apr;286(4):G521-7.

Challenges

Matching the proteome with tag pattern

Developing internal standard for gel and blot

Secondary reactions may also lead to thiol Modification

Thiol proteomes are composed of discreet low abundance proteins

Current Lab MembersAimee Landar

Anne DiersYeun Su ChooKarina Ricart

Michelle Johnson Stephen Barnes

Paul BrookesDale Dickinson Jason MorrowLewis Pannell

Shannon BaileyNeil Hogg

Scott Ballinger

Philip EatonBruce King

Elena UlasovaJoo-Yeun Oh

Jessica GutierrezBrian DrankaBalu Chacko

Ashlee PrestonJeff Dubuisson

Former MembersNobuo Watanabe

Jaroslaw Zmijewski Claire Le Goffe Niroshini Giles

Anna-Liisa LevonenSruti Shiva

Collaborators

Selected References for Thiol

Proteomics • Eaton, P. (2006) Protein thiol oxidation in health and disease: techniques for measuring disulfides and related

modifications in complex protein mixtures. Free Radic Biol Med 40, 1889-1899• Good overview of the various methods available for measuring thiol redox status in a proteomics context and the

principles involved. • Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M. and King, S. B. (2005) Synthesis of chemical probes to map sulfenic

acid modifications on proteins. Bioconjug Chem 16, 1624-16028.• Example of the strategies to develop a thiol tag that can be applied to proteomics.• Landar, A., Oh, J. Y., Giles, N. M., Isom, A., Kirk, M., Barnes, S. and Darley-Usmar, V. M. (2006) A sensitive method for the

quantitative measurement of protein thiol modification in response to oxidative stress. Free Radic Biol Med 40, 459-468• Method for the quantitative measurement of biotin tags in proteomics gel formats. • Patton, W. F. (2002) Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci 771, 3-

31• Broad overview of the various approaches to assessing post-translational modification of proteomes. • Gao, C., Guo, H., Wei, J., Mi, Z., Wai, P. Y. and Kuo, P. C. (2005) Identification of S-nitrosylated proteins in endotoxin-

stimulated RAW264.7 murine macrophages. Nitric Oxide 12, 121-126.• An application of the biotin switch method as applied to S-nitrosothiols showing endogenous protein S-nitrosation. • Gladwin, M. T., Wang, X. and Hogg, N. (2006) Methodological vexation about thiol oxidation versus S-nitrosation -- a

commentary on "An ascorbate-dependent artifact that interferes with the interpretation of the biotin-switch assay". Free Radic Biol Med 41, 557-561

• Discussion of the problem of false positives in biotin switch methods.• Dennehy, M. K., Richards, K. A., Wernke, G. R., Shyr, Y. and Liebler, D. C. (2006) Cytosolic and nuclear protein targets of

thiol-reactive electrophiles. Chem Res Toxicol 19, 20-29• Use of mass spectrometry proteomics analysis to define the electrophile responsive proteome in cells. • Levonen, A. L., Landar, A., Ramachandran, A., Ceaser, E. K., Dickinson, D. A., Zanoni, G., Morrow, J. D. and Darley-

Usmar, V. M. (2004) Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products. Biochem J 378, 373-382

• An example of the candidate protein approach using different tagging approaches to identify modification of a cell signaling molecule.