Figure 2. RSNO capture by the triphenylphosphine-thioesters yields disulfide-iminophosphorane.

Trapping S-nitrosothiols by “SNOTRAP”

Reference: Seneviratne et al., J. Am. Chem. Soc. 2013, 135 (20), 7693-7704.

Introduction

Decoding the S-nitroso Proteome in a Transgenic Mouse Model of Alzheimer’s by SNOTRAP and Mass Spectrometry – Clues for Altered Signaling Pathways

Uthpala Seneviratne1, Alexi Nott2,3, , Ravindra Kodihalli1, Vadiraja Bhat4, John S. Wishnok1, Li-Huei Tsai2,3 and Steven R. Tannenbaum1,5 Agilent Technologies Inc.4 Wilmington , DE. Departments of Biological Engineering1, The Picower Institute for Learning and Memory2, Brain and Cognitive Sciences3 and Chemistry5, Massachusetts Institute of Technology, Cambridge, MA

Methods and Results

Ph2PS

NX

O

Ph2PN SR

S

O

XPh2P

S

N SR

XO

SR

azaylide disulfide-iminophosporane

PPh2S

O

S-nitrosothiolsRSNO

B

B

BB

B = Biotin

SNOTRAP

Figure 1. S-nitrosation plays a dynamic role in neuronal signal transduction pathways and disease progression.

Nitric oxide (NO•) is produced by three nitric oxide synthase isoforms (nNOS, eOS2 and iNOS) at low levels as a signaling molecule and at higher concentrations in pathophysiological conditions. NO• reacts with glutathione (GSH) to form a S-nitrosothiol of glutathione (GSNO) and then is available for transnitrosation to cysteine residues on proteins (PSNOs), as a reversible post-translational modifications (PTM) to regulate the activities of enzymes in key biochemical pathways.

Ca2+

NMDAR

Ca2+

nNOS

CaM Ca2+

Ca2+

NO

Ca2+

Ca2+ Ca2+ Ca2+

O2-�

ONOO-

DNA damage (DSBs) due to Oxidative and nitrosative stress

�OH

Protein-SNO

Signal modulation Pathology

iNOS

GS�

GSNO

sGC cGMP

Protein nitration

•  Aβ oligomers •  Environmental toxins •  Aggregated/misfolded proteins •  Neuroinflammatory stimuli •  Aging

Transnitrosation

Figure 5. Proteomic workflows for the SNO-protein ID and SNO-site ID.

50 µm

A high throughput proteomic approach for target identification of global S-nitrosation

Adapted and modified from, Nakamura et al. Neuron. 2013 (78), 596-614

PPh2S

O

O NH3

O

SNH

HN O

SNOTRAP reagent for proteomics

HS

SNO IAM H2N

OI

SNOTRAP

S

O3

NH

O

SNH

HN O

P N

OPh2SNO

S S

S

1.  Trypsin digestion 2.  Streptavidin enrichment

1. TCEP 2. NEM

S N

O

O

S S

Stre

ptav

idin

Bio

tin

3 x10

0

0.5

1

1.5

2

2.5

3

m/z, Da 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

944.46786 (M2+)

Figure 6. Use of Diagnostic fragment ions (DFIs) for the SNO-site identification in brain homogenate, Cys-73 of TRX1, Cys-47 of PRDX6 and Cys-150 of GAPDH.

Site-specific mapping of protein S-nitrosation v  We found that the NEM modification on cysteine produced several major diagnostic fragment ions (DFIs) at low

mass region, m/z = 158.0276, 126.0550, a neutral loss of 125.0477, which are generated by fragmentations analogous to those reported for cysteine conjugates.

MS/MS (CID)

y10 y9 y102+

y4

y13 a2

y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1

I V S N A S *C T T N C L A P L A K

b2 y2

y5

y9-H2O2+ b13

b6-H2O2+

b13-H2O

y12

y11

y152+

y142+

y15-H2O2+

0

m/z 40 60 80 100 120 140 160 180 200 220

125.0485 DFI-3

a2

b2 y2

126.0542 DFI-1

3 x10

0

0.2

0.4

0.6

0.8

1

1.2

m/z, Da 100 200 300 400 500 600 700 800 900 1000 1100 1200

72.08163

A

B

C

Thioredoxin (73-81)

4 x10

0

1

2

3

4

5

6

7

8

m/z, Da 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

b1 b2 b3

y7 y6 y5 y4 y3 y2 y1

*C M P T F Q F F K

y7 y6 y5 y4

y3 y2 y1

y10 y8

y102+

y5

b3

b2

b2 b3

0 0.5

1 1.5

2 2.5

3 3.5

4 4.5

5

m/z 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180

126.0549 158.0250

DFI-2

y1

DFI-1

Peroxiredoxin-6, PRDX-6 (42-53)

b1 b2 b3

y10 y9 y8 y7 y6 y5 y4 y3 y2 y1

D F T P V *C T T E L G R

y9

y7

y92+

y10-H2O2+

DFI-2

GAPDH (144-160)

Figure 3. Differencial S-nitrosation levels in postnatal (P2) mouse brain (left) and in E17 and P2 mice cortex (right).

Mapping the mouse brain for endogenous GSNO (extent of S-nitrosation)

[GSNO] ± SD µM Cerebral Cortex Embryonic 0.7 ± 0.2

Postnatal day 2 0.4 ± 0.1

Spaciotemporal changes in GSNO levels in Cdk5/p25 mouse brain; Clues for nitrosative stress

Figure 4. Higher GSNO levels were observed in hippocampus of 2 week Cdk5/p25 mouse (n = 4).

0.6 ± 0.07 µM

0.4 ± 0.06 µM

1.1 ± 0.1 µM

mid brain 0.7 ± 0.07 µM

0.9 ± 0.1 µM

P2 mouse brain

0

0.2

0.4

0.6

0.8

E17 P2

[GSN

O] µ

M

Extent of S-nitrosation Embryonic vs Postnatal GSNO in Cerebral Cortex of E17 Vs p2 (n=6)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Ctrl CKp25 Ctrl CKp25 Ctrl CKp25 Ctrl CKp25 Ctrl CKp25 Ctrl CKp25

Cerebral Cortex Hippocampus

Cerebellum

[GSN

O] µ

M Increase in NO

production, Nitrosative

stress !!

Weeks after p25 induction 2 4 5-6 8 27

DNA damage Astrogliosis

Neuronal/synaptic loss

Learning impairment

Insoluble Tau Neurofibrillary tangle

pathology

Diagnostic Fragment ions (DFIs)

H2N

O

H2N

O

HN NH

O

S

NO

O

m/z = 158.0276, 176.0381 (+ H2O)

m/z = 126.0550, 144.0661 (+ H2O)

NO

O

Neutral loss: 125.0477

Experimental design

0

m/z 60 80 100 120 140 160 180 200 220

72.0816

125.0473

158.0432

DFI-3

Acknowledgements This work was supported by the NIH (CA26731), Simons Center For The Social Brain at MIT and by the MIT Center for Environmental Health Sciences (ES002109). We acknowledge Agilent Technologies for access to the UHPLC 1290 binary pump, autosampler, and the QqQ-MS (6430). We thank Thakshila Dissanayake for assistance with developing the in silico filter algorithm for peptide ID.

Identification >300 S-nitrosated proteins in the cortex and hippocampus in 2-week Cdk5/p25 mice

Identified >400 proteins, including the site of S-nitrosation in Cortex, Hippocampus and Cerebellum, by Agilent 6550 Qtof MS.

KEGG and PANTHER Pathway analysis of the Cortex and Hippocampus of Cdk5/p25 mouse model

Functional analysis of S-nitroso proteome (S-nitrosylome) in Cdk5/p25 mouse model of neurodegeneration

Figure 7. Bioinformatics analysis of the S-nitrosylome.

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Cell adhesion molecules (CAMs)

Cardiac muscle contraction

Calcium signaling pathway

Long-term potentiation

Alzheimer's disease

Aldosterone-regulated sodium reabsorption

Phosphatidylinositol signaling system

Axon guidance

Inositol phosphate metabolism

Gap junction

Prion diseases

Dilated cardiomyopathy

Melanogenesis

Focal adhesion

Endocytosis

Tight junction

Arrhythmogenic right ventricular cardiomyopathy

Log10 P

v  Alzheimer disease-amyloid secretase pathway

v  Alzheimer disease-presenilin pathway

PKC

Protein targets of S-nitrosation in Alzheimer’s disease, ID by SNOTRAP in Cdk5/p25 mouse model

Ctrl mouse Linear Sequence Motif Analysis by pLogo

More basic/nucleophilic amino acids

Full motif gets nitrosated at physiological conditions

Partial motif gets nitrosated when high NO (GSNO) conditions

Cdk5/p25 mouse

Acidic & basic amino acids

0 2 4 6 8 10 12

cellular process cell adhesion

biological adhesion cell communication

nervous system development neurological system process

synaptic transmission transport

localization transcription, DNA-dependent

cell-cell signaling transcription from RNA polymerase II promoter

regulation of transcription from RNA polymerase ion transport

ellular protein modification process RNA metabolic process

system process cation transport

protein phosphorylation regulation of nucleobase-containing compound

protein metabolic process ectoderm development

Catalytic activity Protein kinase activity

Cation transmembrane transporter activity DNA binding

Log10 P

GO annotations (Biological Process and Molecular Function) enriched in S-nitrosylome (in mouse brain by SNOTRAP)

Negative controls were generated by, ²  Cu/ascorbate ²  TCEP/DTT ²  UV light (312 nm) followed by IAM and SNOTRAP.

Samples were processed by FASP, a proteomic MWCO

reactor, FASP; filter-aided sample prep,

Nat. Methods 2009, 6, 359

DNA damage was observed in 2 week mouse

Hippocampus CA1 region

double strand breaks (DSBs)

β-Amyloid increase

1 µm

Weeks after p25 induction

2 wks 6 wks

DNA damage Neuronal/synaptic loss Learning impairment

Control CK-p25 Control CK-p25

Mass Spectrometry Analysis

Western blot Analysis

Bioinformatics – functional clustering & network approaches

3 brain regions, 2 time points

Kim and Tsai et al. Neuron. 2008 (60), 803-817

KEGG Pathways

CKp25

Ctrl

164 60%

34 16%

62 24%

Cerebral Cortex

CKp25 Ctrl

38 28%

27 20%

68 52%

Cerebellum CKp25

Ctrl

58 60%

23 23%

17 17%

Hippocampus

By PANTHERdb.org

We present here a novel methodology, termed “SNOTRAP” (SNO trapping by TriAryl Phosphine), a phosphine ligation strategy for specific detection of SNO functionality, where SNOs were selectively targeted based on their distinct chemical reactivity profile and converted to a more stable disulfide-iminophosphorane by a Staudinger-type reductive ligation.

The proteomic workflow allows the multiplexing by TMT tags, enabling quantification of relative abundance of the cysteine modified peptide.

2 week 6 week

SNOS SSH SNOS SS

SNOTRAP

SS SSS

Trypsin digest

Blocking; Alkylation Cys with IAM;

carbamidomethylTissue homogenate

SS SSS

SS SSSS

S

1. Wash2. TCEP reductive elution3. Alkylation with NEM4. C-18 Desalt

SNEM

Protein ID and SNO-site ID

m/z

1. Wash 2. SDS/DTT3. Gel-electrophoresis

Western blot

steptavidin-agarose

steptavidin-agarose

30 KDa Protein ID

Western blot

1. Wash 2. Elution (ACN/TFA)

3. Gel-electrophoresis4. Streptavidin-HRP

nLC-MS/MS

Data-base search