SNOB Technology
The Company has developed a NOVEL and patented single step labelling assay for the visual detection and proteomic characterisation of protein S-nitrosylation. Its founding scientists, Drs Andrew Watts and Amanda Mackenzie are specialist chemists and biologists in this field. The assay is suitable for use in live cells and protein lysates, and amenable to manufacture for sale in kit format. S-Nitrosylation is highly relevant to inflammatory disease and apoptosis and this technology will be of interest to both pharmaceutical and academic researchers in the fields. The assay is currently available in a biotin format for protein lysates and proteomic characterisation, while a fluorescent version is being developed for live cell microscopy experiments.
There is a patent currently pending and licences would be available for those wishing to use patented technology in a service environment for the biopharmaceutical and pharmaceutical industries.
Purchase SNOB Reagents/Kits from Tocris Bioscience
Glythera has recently granted Tocris Bioscience an exclusive license to supply SNOB reagents and/or kits for use solely as preclinical research compounds. Accordingly, any researchers wishing to obtain authentic SNOB products are invited to visit the Tocris Bioscience website for further information.
Background:
Nitric oxide (NO) is a ubiquitous signalling molecule that, in part, mediates its actions via direct protein modification. Reversible addition of NO to target cysteine thiol to form an S-nitrosothiol group (SNO), a process termed S-nitrosylation, can modify the function or activity of a broad range of proteins. The action of direct protein nitrosylation or denitrosylation as a cellular signalling pathway is poorly understood, although nitrosylation is known to play a role in disease processes such as inflammation, apoptosis and ischemiareperfusion injury. It is expected that S-nitrosylation will emerge as a highly relevant protein modification similar to phosphorylation in cell signalling. Important physiological roles for S-nitrosylation include inhibition of NMDA receptor activation under hypoxic conditions, regulation of G-protein-coupled receptor kinase 2 activity and activation of TRPC5 channels. The Company has developed a single-step assay which directly detects endogenous S-nitrosylation of cysteine residues and provides clear advantages and improvements over the multi-step ‘biotin-switch’ protocol which is the current method of choice for detecting S-nitrosylation.
Mode of action:
The SNO-Binding (SNOB) reagents directly and specifically label S-nitrosothiol groups on proteins in a single chemical step which is compatible with physiological conditions. SNOB reagents take advantage of the inherent chemical orthoganality and reactivity of the S-NO bond, and in doing so by-pass the need for the multiple chemical steps required by the Biotin Switch method. This new approach allows direct detection and functionalization of endogenously S-nitrosylated proteins, enabling greater exploration into the physiological roles of NO. Moreover, the single-step labelling of SNO groups under physiological conditions will facilitate the visualisation of S-nitrosylation profiles in living systems using a variety of molecular imaging techniques.
Figure 1. Representation showing a SNOB reagent reacting with an S-nitrosylated protein.
Key Features:
1. Visualisation of Endogenous S-nitrosylation in acutely isolated Human platelets
Endogenous S-nitrosylated proteins have been detected on the cell surface of acutely isolated human platelets. Using this approach, isolated platelets were incubated with SNOB (450 ìM) reagent (5 minutes) either in the presence or absence of an NO donor or dithiothreitol (DTT, 20 mM) at 37 ºC. Following incubation with SNOB, platelets were isolated by centrifugation that will also remove excess SNOB reagents and protein biotinylation detected by SDS-PAGE and Western blot using neutravidin HRP to detect biotin groups. Protein biotinylation was detected in platelets (in the absence of an NO donor) and reduced by incubation with DTT expected to remove SNO groups from proteins (Figure 2). Incubation of platelets with a NO donor increased protein biotinylation above that detected in untreated platelets. This technique represents a novel approach to detect cell surface S-nitrosylation in live cells at 37 ºC.
Figure 2. Direct visual detection of endogenous S-Nitrosylation in Human platelets using SNOB reagent.
2. Proteomic characterisation of S-nitrosylated Bovine serum albumin:
We have used SNOB reagents to detect and characterise S-nitrosylation of commercial BSA using mass spectroscopy. BSA protein contains 35 cysteine residues, 34 of which are involved in disulphide bridges, with only one surface cysteine (Cys 58) available for nitrosylation. A sample of nitrosylated (DEANOnoate) BSA (50 ìL, 1 mg/mL) was treated with SNOB reagent (450ìM) for 5 minutes at room temperature (20mM Tris-HCl, pH 7). The mixture was then subjected to proteolytic digestion with pepsin (0.5 mL, 1 mg/mL, 1M phosphate, pH 2) for 4 hours at room temperature. The sample was then purified (Neutravidin beads, pH 10) and analysed by LC/MS. A single peptide fragment QQCP+ SNOB linker was observed as both the +H and +Na ion (Figure 3).
This labelled peptide fragment contains the single surface cysteine residue (Cys 58) of BSA, and most importantly, incorporates a carbonyl oxygen derived from the original NO group, providing unambiguous confirmation that labelling has occurred at a nitrosylated cysteine residue.
Figure 3. A. Mass spectrum of nitrosylated BSA following labelling with SNOB and pepsin digestion. Single peptide fragment is detected. B. Sequence of detected peptide corresponds to amino acid sequence incorporating single surface cysteine QQCP+ SNOB linker.
Patent Information:
For details of our patent application - click here
References:
Andrew G Watts and Amanda B MacKenzie, Detection and Functionalisation of S-nitrosylated Polypeptides. Intl. Patent Appltn. PCT/GB2008/002838, (21-08-2007) Patent Pending.
Nott, A., Watson, P.M., Robinson, J.D., Crepaldi, L., Riccio, A. (2008) S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons Nature 455, 411 - 415
Tada Y, Spoel SH, Pajerowska-Mukhtar K, Mou Z, Song J, Wang C, Zuo J, Dong X. (2008) Plant Immunity Requires Conformational Charges of NPR1 via S-Nitrosylation and Thioredoxins. Science 321, 952-956
Whalen EJ, Foster MW, Matsumoto A, Ozawa K, Violin JD, Que LG, Nelson CD, Benhar M, Keys JR, Rockman HA, Koch WJ, Daaka Y, Lefkowitz RJ, Stamler JS. (2007) Regulation of E-Adrenergic Receptor Signaling by S-Nitrosylation of G-Protein-Coupled Receptor Kinase 2. Cell 129, 511 - 522
Takahashi H, Shin Y, Cho SJ, Zago WM, Nakamura T, Gu Z, Ma Y, Furukawa H, Liddington R, Zhang D, Tong G, Chen HS, Lipton SA. (2007) Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif. Neuron 53(1):53-64.
The Company has developed a NOVEL and patented single step labelling assay for the visual detection and proteomic characterisation of protein S-nitrosylation. Its founding scientists, Drs Andrew Watts and Amanda Mackenzie are specialist chemists and biologists in this field. The assay is suitable for use in live cells and protein lysates, and amenable to manufacture for sale in kit format. S-Nitrosylation is highly relevant to inflammatory disease and apoptosis and this technology will be of interest to both pharmaceutical and academic researchers in the fields. The assay is currently available in a biotin format for protein lysates and proteomic characterisation, while a fluorescent version is being developed for live cell microscopy experiments.
There is a patent currently pending and licences would be available for those wishing to use patented technology in a service environment for the biopharmaceutical and pharmaceutical industries.
Purchase SNOB Reagents/Kits from Tocris Bioscience
Glythera has recently granted Tocris Bioscience an exclusive license to supply SNOB reagents and/or kits for use solely as preclinical research compounds. Accordingly, any researchers wishing to obtain authentic SNOB products are invited to visit the Tocris Bioscience website for further information.
Background:
Nitric oxide (NO) is a ubiquitous signalling molecule that, in part, mediates its actions via direct protein modification. Reversible addition of NO to target cysteine thiol to form an S-nitrosothiol group (SNO), a process termed S-nitrosylation, can modify the function or activity of a broad range of proteins. The action of direct protein nitrosylation or denitrosylation as a cellular signalling pathway is poorly understood, although nitrosylation is known to play a role in disease processes such as inflammation, apoptosis and ischemiareperfusion injury. It is expected that S-nitrosylation will emerge as a highly relevant protein modification similar to phosphorylation in cell signalling. Important physiological roles for S-nitrosylation include inhibition of NMDA receptor activation under hypoxic conditions, regulation of G-protein-coupled receptor kinase 2 activity and activation of TRPC5 channels. The Company has developed a single-step assay which directly detects endogenous S-nitrosylation of cysteine residues and provides clear advantages and improvements over the multi-step ‘biotin-switch’ protocol which is the current method of choice for detecting S-nitrosylation.
Mode of action:
The SNO-Binding (SNOB) reagents directly and specifically label S-nitrosothiol groups on proteins in a single chemical step which is compatible with physiological conditions. SNOB reagents take advantage of the inherent chemical orthoganality and reactivity of the S-NO bond, and in doing so by-pass the need for the multiple chemical steps required by the Biotin Switch method. This new approach allows direct detection and functionalization of endogenously S-nitrosylated proteins, enabling greater exploration into the physiological roles of NO. Moreover, the single-step labelling of SNO groups under physiological conditions will facilitate the visualisation of S-nitrosylation profiles in living systems using a variety of molecular imaging techniques.
Figure 1. Representation showing a SNOB reagent reacting with an S-nitrosylated protein.
Key Features:
- Single step labelling of SNO to generate stable thioether bond and labelled NO group
- Direct detection of endogenously S-nitrosylated proteins
- Compatible over a wide range of conditions ( 4 - 60°C, pH 3 - 11, reducing conditions)
- Works in both live cells and protein lysates
- Variety of detection methods including biotin/streptavidin conjugates, fluorescence and mass spec
- Single step compared to the multistep ‘biotin-switch’ technique
- No need for thiol blocking step
- Reagent reacts directly with SNO to generate a stable thioether linked product for detection
- Lower background noise
- More elegant and direct chemistry leads to higher reproducibility and sensitivity
- Works in living cells and on protein lysates in vitro whereas biotin switch only detects denatured proteins in cell lysates and relies on the stability of the SNO group
1. Visualisation of Endogenous S-nitrosylation in acutely isolated Human platelets
Endogenous S-nitrosylated proteins have been detected on the cell surface of acutely isolated human platelets. Using this approach, isolated platelets were incubated with SNOB (450 ìM) reagent (5 minutes) either in the presence or absence of an NO donor or dithiothreitol (DTT, 20 mM) at 37 ºC. Following incubation with SNOB, platelets were isolated by centrifugation that will also remove excess SNOB reagents and protein biotinylation detected by SDS-PAGE and Western blot using neutravidin HRP to detect biotin groups. Protein biotinylation was detected in platelets (in the absence of an NO donor) and reduced by incubation with DTT expected to remove SNO groups from proteins (Figure 2). Incubation of platelets with a NO donor increased protein biotinylation above that detected in untreated platelets. This technique represents a novel approach to detect cell surface S-nitrosylation in live cells at 37 ºC.
Figure 2. Direct visual detection of endogenous S-Nitrosylation in Human platelets using SNOB reagent.
2. Proteomic characterisation of S-nitrosylated Bovine serum albumin:
We have used SNOB reagents to detect and characterise S-nitrosylation of commercial BSA using mass spectroscopy. BSA protein contains 35 cysteine residues, 34 of which are involved in disulphide bridges, with only one surface cysteine (Cys 58) available for nitrosylation. A sample of nitrosylated (DEANOnoate) BSA (50 ìL, 1 mg/mL) was treated with SNOB reagent (450ìM) for 5 minutes at room temperature (20mM Tris-HCl, pH 7). The mixture was then subjected to proteolytic digestion with pepsin (0.5 mL, 1 mg/mL, 1M phosphate, pH 2) for 4 hours at room temperature. The sample was then purified (Neutravidin beads, pH 10) and analysed by LC/MS. A single peptide fragment QQCP+ SNOB linker was observed as both the +H and +Na ion (Figure 3).
This labelled peptide fragment contains the single surface cysteine residue (Cys 58) of BSA, and most importantly, incorporates a carbonyl oxygen derived from the original NO group, providing unambiguous confirmation that labelling has occurred at a nitrosylated cysteine residue.
Figure 3. A. Mass spectrum of nitrosylated BSA following labelling with SNOB and pepsin digestion. Single peptide fragment is detected. B. Sequence of detected peptide corresponds to amino acid sequence incorporating single surface cysteine QQCP+ SNOB linker.
Patent Information:
For details of our patent application - click here
References:
Andrew G Watts and Amanda B MacKenzie, Detection and Functionalisation of S-nitrosylated Polypeptides. Intl. Patent Appltn. PCT/GB2008/002838, (21-08-2007) Patent Pending.
Nott, A., Watson, P.M., Robinson, J.D., Crepaldi, L., Riccio, A. (2008) S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons Nature 455, 411 - 415
Tada Y, Spoel SH, Pajerowska-Mukhtar K, Mou Z, Song J, Wang C, Zuo J, Dong X. (2008) Plant Immunity Requires Conformational Charges of NPR1 via S-Nitrosylation and Thioredoxins. Science 321, 952-956
Whalen EJ, Foster MW, Matsumoto A, Ozawa K, Violin JD, Que LG, Nelson CD, Benhar M, Keys JR, Rockman HA, Koch WJ, Daaka Y, Lefkowitz RJ, Stamler JS. (2007) Regulation of E-Adrenergic Receptor Signaling by S-Nitrosylation of G-Protein-Coupled Receptor Kinase 2. Cell 129, 511 - 522
Takahashi H, Shin Y, Cho SJ, Zago WM, Nakamura T, Gu Z, Ma Y, Furukawa H, Liddington R, Zhang D, Tong G, Chen HS, Lipton SA. (2007) Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif. Neuron 53(1):53-64.