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(20) Identification of organophosphate-reactive proteins by tandem mass spectrometry
Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434
383
(20) Identification of organophosphate-reactive proteins by tandem mass spectrometry He Li a , Lawrence Schopfer a, Reggie Spaulding b, Charles M. Thompson b, Oksana Lockridge a a
Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-18105, USA b Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 598121552, USA 1. Introduction Organophosphorus (OP) compounds have been used as therapeutic agents, agricultural chemicals and as chemical warfare agents. The toxicity of OP compounds is initiated by inhibition of acetylcholinesterase (AChE) in the central and peripheral nervous systems. Despite the fact that AChE is the primary target of OPs, there is evidence indicating that OPs also interact with other proteins and these interactions have physiological significance [1,2]. Serine hydrolases play important roles in numerous developmental and tissue-specific events in vivo. The majority of serine hydrolases have been shown to be potently and irreversibly inhibited by fluorophosphonate (FP) derivatives. In this study, we used a biotinylated fluorophosphonate (FPB) probe to label and subsequently separate the OP reactive targets from mouse brain. In the last few years, mass spectrometry has become an increasingly powerful and indispensable technology to identify the primary structure and post-translational modifications of proteins. Mass spectrometry was applied as the central tool in identifying the OP reactive proteins in this study. 2. Experimental procedures and results A custom synthesized fluorophosphonate (FP)-biotin compound (MW = 593) was used to label the OP reactive proteins in mouse brain 100,000 × g supernatant. The labeling reaction was carried out at 25 ◦ C for 5 h to ensure that all OP reactive proteins were labeled. The labeled proteins were extracted by biotin binding to Avidin-agarose beads. We separated the labeled proteins by running the beads on a 10–20% SDS–PAGE gel. A total of 55 bands were visualized on the gel after 6 h of Coomassie blue G-250 staining (Fig. 1). Each visible band was sliced out as gel strip and subjected to in-gel trypsin digestion. The tryptic peptides generated from overnight digestion was extracted from the gel bits by addition of 200 ul of 0.1% TFA in 60%
Fig. 1. SDS–PAGE separation of FPB labeled proteins.
acetonitrile. They were ready for mass spectrometry analysis. A Waters CapLC capillary liquid chromatography system was coupled to a Micromass quadrupole/timeof-flight (Q-TOF) tandem mass spectrometer for the analysis of tryptic peptides. Peptides in aliquot were separated on a C18 reverse phase column by a gradient with increasing organic solvent concentration. Peptides eluted from the column were introduced into the mass spectrometer as positively charged particles. MS spectra (i.e. the precursor ion spectra) were acquired throughout the gradient run; the subsequent MS/MS spectra (i.e. the product ion spectra) were acquired in a data dependent fashion: the three most abundant precursor ions identified in any given MS spectrum would be captured and broken up to generate their MS/MS spectrum respectively. The MS/MS spectrum is unique to each peptide based on its amino acid sequence, thus the peptides can be identified and further the protein to which it belongs. All MS/MS spectra were searched against the SwissProt database using MASCOT program. The search criteria were set with precursor ions mass accuracy of ±1.0 Da and product ions mass accuracy of ±0.5 Da. One missed cleavage by trypsin was allowed. MASCOT calculates a score for each identified protein based on the match between the experimental peptide mass and the theoretical peptide mass, as well as between the experimental MS/MS spectra and the theoretical fragment ions from each peptide. 3. Conclusion A total of 28 proteins were identified with high sequence coverage. Eighteen of them are hydrolases, including acylpeptide hydrolase, prolylendopeptidase
Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434
384
Table 1 Summary of MASCOT analysis of tandem MS/MS data Band #
SDS–PAGE MW kDa
% Total protein found
Protein name (mouse proteins only)
5 6 7 8 12 17 18 23 35 38 41 44/45 50 53
110 100 95 90 85 75 65 60 40 35 30 25 20 20
18 4 15 10 26 9 3 7 21 23 27 56 20 31
Pyruvate carboxylase/decarboxylase Puromycin-sensitive aminopeptidase Formyltetrahydrofolate dehydrogenase Heat shock protein 90-Beta Prolylendopeptidase Serum albumin precursor Protein phosphatase 2 D-3-phosphoglycerate dehydrogenase Acyl-CoA hydrolase Glyceraldehyde-3-phosphate dehydrogenase Lactate dehydrogenase 1 Platelet-activating factor acetylhydrolase alpha-1 Lysophospholipase 1 Peroxiredoxin 1
and protein phosphatase 2 (Table 1). The result indicates a broad range of potential OP reactive proteins in mouse brain, among which hydrolases are the major targets. AChE and BChE were not identified in this experiment because the low abundance of these two proteins in mouse brain made these bands too weak to be seen with Coomassie blue staining. The significance of the reaction between these proteins and OP needs to be further characterized. Mass spectrometry proved to be a potent way for identification of target proteins from a complex mixture in this study. Acknowledgement This work was supported by U.S. Army Medical Research and Materiel Command DAMD 17-01-2-0036 and DAMD 17-01-1-0776. References [1] C.N. Pope, Organophosphorus pesticides: do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health B Crit. Rev. 2 (2) (1999) 161–181. [2] E.G. Duysen, B. Li, W. Xie, L.M. Schopfer, R.S. Anderson, C.A. Broomfield, O. Lockridge, Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to VX lethality, J. Pharmacol. Exp. Ther. 299 (2) (2001) 528–535.
doi:10.1016/j.cbi.2005.10.065
(21) Measuring carbamoylation and decarbamoylation rate constants by continuous assay of AChE Joseph L. Johnson, Jamie L. Thomas , Sujata Emani , Bernadette Cusack, Terrone L. Rosenberry ∗ Mayo Clinic College of Medicine, Departments of Neuroscience and Pharmacology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA Keywords: Acetylcholinesterase; Carbamoylation; Substrate activation The active site gorge of acetylcholinesterase (AChE) contains two distinct sites of ligand interaction: an acylation site (A-site) at the base of the gorge and a peripheral site (P-site) near the mouth of the gorge at the enzyme surface. While the role of the P-site in catalysis has been elusive, our recent studies have shown that it serves as an intermediate binding site for cationic ligands as they proceed to the A-site. P-site binding also can lead to allosteric activation of the acylation step and/or to steric blockade, defined as an equal reduction in the association and dissociation rate constants for the binding of a second ligand to the A-site when the P-site is occupied. Compelling evidence that allosteric activation occurs at the acylation step came from examination of the hydrolysis of a close substrate analog of acetylcholine, the acetanilide ATMA [1]. The acylation step in the AChE hydrolysis of ATMA can be isolated because of the slow cleavage of the anilide bond, revealing the positive deviation from Michaelis-Menten kinetics that defines substrate activation. However, not all substrates that bind to the P-site show allosteric activation of acylation. In fact,
383
(20) Identification of organophosphate-reactive proteins by tandem mass spectrometry He Li a , Lawrence Schopfer a, Reggie Spaulding b, Charles M. Thompson b, Oksana Lockridge a a
Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-18105, USA b Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 598121552, USA 1. Introduction Organophosphorus (OP) compounds have been used as therapeutic agents, agricultural chemicals and as chemical warfare agents. The toxicity of OP compounds is initiated by inhibition of acetylcholinesterase (AChE) in the central and peripheral nervous systems. Despite the fact that AChE is the primary target of OPs, there is evidence indicating that OPs also interact with other proteins and these interactions have physiological significance [1,2]. Serine hydrolases play important roles in numerous developmental and tissue-specific events in vivo. The majority of serine hydrolases have been shown to be potently and irreversibly inhibited by fluorophosphonate (FP) derivatives. In this study, we used a biotinylated fluorophosphonate (FPB) probe to label and subsequently separate the OP reactive targets from mouse brain. In the last few years, mass spectrometry has become an increasingly powerful and indispensable technology to identify the primary structure and post-translational modifications of proteins. Mass spectrometry was applied as the central tool in identifying the OP reactive proteins in this study. 2. Experimental procedures and results A custom synthesized fluorophosphonate (FP)-biotin compound (MW = 593) was used to label the OP reactive proteins in mouse brain 100,000 × g supernatant. The labeling reaction was carried out at 25 ◦ C for 5 h to ensure that all OP reactive proteins were labeled. The labeled proteins were extracted by biotin binding to Avidin-agarose beads. We separated the labeled proteins by running the beads on a 10–20% SDS–PAGE gel. A total of 55 bands were visualized on the gel after 6 h of Coomassie blue G-250 staining (Fig. 1). Each visible band was sliced out as gel strip and subjected to in-gel trypsin digestion. The tryptic peptides generated from overnight digestion was extracted from the gel bits by addition of 200 ul of 0.1% TFA in 60%
Fig. 1. SDS–PAGE separation of FPB labeled proteins.
acetonitrile. They were ready for mass spectrometry analysis. A Waters CapLC capillary liquid chromatography system was coupled to a Micromass quadrupole/timeof-flight (Q-TOF) tandem mass spectrometer for the analysis of tryptic peptides. Peptides in aliquot were separated on a C18 reverse phase column by a gradient with increasing organic solvent concentration. Peptides eluted from the column were introduced into the mass spectrometer as positively charged particles. MS spectra (i.e. the precursor ion spectra) were acquired throughout the gradient run; the subsequent MS/MS spectra (i.e. the product ion spectra) were acquired in a data dependent fashion: the three most abundant precursor ions identified in any given MS spectrum would be captured and broken up to generate their MS/MS spectrum respectively. The MS/MS spectrum is unique to each peptide based on its amino acid sequence, thus the peptides can be identified and further the protein to which it belongs. All MS/MS spectra were searched against the SwissProt database using MASCOT program. The search criteria were set with precursor ions mass accuracy of ±1.0 Da and product ions mass accuracy of ±0.5 Da. One missed cleavage by trypsin was allowed. MASCOT calculates a score for each identified protein based on the match between the experimental peptide mass and the theoretical peptide mass, as well as between the experimental MS/MS spectra and the theoretical fragment ions from each peptide. 3. Conclusion A total of 28 proteins were identified with high sequence coverage. Eighteen of them are hydrolases, including acylpeptide hydrolase, prolylendopeptidase
Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434
384
Table 1 Summary of MASCOT analysis of tandem MS/MS data Band #
SDS–PAGE MW kDa
% Total protein found
Protein name (mouse proteins only)
5 6 7 8 12 17 18 23 35 38 41 44/45 50 53
110 100 95 90 85 75 65 60 40 35 30 25 20 20
18 4 15 10 26 9 3 7 21 23 27 56 20 31
Pyruvate carboxylase/decarboxylase Puromycin-sensitive aminopeptidase Formyltetrahydrofolate dehydrogenase Heat shock protein 90-Beta Prolylendopeptidase Serum albumin precursor Protein phosphatase 2 D-3-phosphoglycerate dehydrogenase Acyl-CoA hydrolase Glyceraldehyde-3-phosphate dehydrogenase Lactate dehydrogenase 1 Platelet-activating factor acetylhydrolase alpha-1 Lysophospholipase 1 Peroxiredoxin 1
and protein phosphatase 2 (Table 1). The result indicates a broad range of potential OP reactive proteins in mouse brain, among which hydrolases are the major targets. AChE and BChE were not identified in this experiment because the low abundance of these two proteins in mouse brain made these bands too weak to be seen with Coomassie blue staining. The significance of the reaction between these proteins and OP needs to be further characterized. Mass spectrometry proved to be a potent way for identification of target proteins from a complex mixture in this study. Acknowledgement This work was supported by U.S. Army Medical Research and Materiel Command DAMD 17-01-2-0036 and DAMD 17-01-1-0776. References [1] C.N. Pope, Organophosphorus pesticides: do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health B Crit. Rev. 2 (2) (1999) 161–181. [2] E.G. Duysen, B. Li, W. Xie, L.M. Schopfer, R.S. Anderson, C.A. Broomfield, O. Lockridge, Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to VX lethality, J. Pharmacol. Exp. Ther. 299 (2) (2001) 528–535.
doi:10.1016/j.cbi.2005.10.065
(21) Measuring carbamoylation and decarbamoylation rate constants by continuous assay of AChE Joseph L. Johnson, Jamie L. Thomas , Sujata Emani , Bernadette Cusack, Terrone L. Rosenberry ∗ Mayo Clinic College of Medicine, Departments of Neuroscience and Pharmacology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA Keywords: Acetylcholinesterase; Carbamoylation; Substrate activation The active site gorge of acetylcholinesterase (AChE) contains two distinct sites of ligand interaction: an acylation site (A-site) at the base of the gorge and a peripheral site (P-site) near the mouth of the gorge at the enzyme surface. While the role of the P-site in catalysis has been elusive, our recent studies have shown that it serves as an intermediate binding site for cationic ligands as they proceed to the A-site. P-site binding also can lead to allosteric activation of the acylation step and/or to steric blockade, defined as an equal reduction in the association and dissociation rate constants for the binding of a second ligand to the A-site when the P-site is occupied. Compelling evidence that allosteric activation occurs at the acylation step came from examination of the hydrolysis of a close substrate analog of acetylcholine, the acetanilide ATMA [1]. The acylation step in the AChE hydrolysis of ATMA can be isolated because of the slow cleavage of the anilide bond, revealing the positive deviation from Michaelis-Menten kinetics that defines substrate activation. However, not all substrates that bind to the P-site show allosteric activation of acylation. In fact,