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• W2110921177 abstract "Mass spectrometry is a rapid, sensitive, and accurate quantitative approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study the proteolysis of intact viral capsid proteins, the α-glucosidase-catalyzed hydrolysis ofp-nitrophenyl-α-glucopyranoside and the lipoprotein lipase-catalyzed ester hydrolysis of resorufin were examined. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry were used to examine the proteolysis of viral protein capsids, providing information about capsid dynamics and the stabilizing force of viral protein/RNA interactions. In addition,k cat and K m values of enzyme-catalyzed hydrolysis were obtained (without the use of a chromophore). These results also demonstrate the effect an unnatural substrate can have on enzyme activity. Overall, mass spectrometry provides for efficient and quantitative analysis of enzyme-catalyzed reactions, as well as the direct observation of reaction dynamics. Mass spectrometry is a rapid, sensitive, and accurate quantitative approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study the proteolysis of intact viral capsid proteins, the α-glucosidase-catalyzed hydrolysis ofp-nitrophenyl-α-glucopyranoside and the lipoprotein lipase-catalyzed ester hydrolysis of resorufin were examined. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry were used to examine the proteolysis of viral protein capsids, providing information about capsid dynamics and the stabilizing force of viral protein/RNA interactions. In addition,k cat and K m values of enzyme-catalyzed hydrolysis were obtained (without the use of a chromophore). These results also demonstrate the effect an unnatural substrate can have on enzyme activity. Overall, mass spectrometry provides for efficient and quantitative analysis of enzyme-catalyzed reactions, as well as the direct observation of reaction dynamics. electrospray ionization matrix-assisted laser desorption/ionization flock house virus liquid chromatography mass spectrometry The development of efficient methods for monitoring enzymatic activity is important (1.Matayoshi E.D. Wang G.T. Kraft G.A. Erickson J. Science. 1990; 247: 954-958Crossref PubMed Scopus (556) Google Scholar, 2.Siuzdak G. Mass Spectrometry for Biotechnology. Academic Press, San Diego1996Google Scholar, 3.Hunt D.F. Michel H. Dickinson T.A. Shabanowitz J. Cox A.L. Sakaguchi K. Appella E. Grey H.M. Sette A. Science. 1992; 256: 1817-1820Crossref PubMed Scopus (631) Google Scholar, 4.Chait B.T. Wang R. Beavis R.C. Kent S.B. Science. 1993; 262: 89Crossref PubMed Scopus (268) Google Scholar, 5.Miranker A. Robinson C.V. Radford S.E. Dobson C.M. FASEB J. 1996; 10: 93Crossref PubMed Scopus (165) Google Scholar, 6.Kouach M. Belaiche D. Jaquinod M. Couppez M. Kmiecik D. Ricart G. Van Dorsselaer A. Sautiere P. Briand G. Biol. Mass Spectrom. 1994; 23: 283Crossref PubMed Scopus (16) Google Scholar, 7.Krebs J.F. Siuzdak G.H. Dyson J. Stewart J.D. Benkovic S.J. Biochemistry. 1995; 34: 720Crossref PubMed Scopus (36) Google Scholar, 8.Siuzdak G. Krebs J.F. Benkovic S.J. Dyson J.H. J. Am. Chem. Soc. 1994; 116: 7937Crossref Scopus (27) Google Scholar, 9.Ganem B. Li Y.T. Henion J.D. J. Am. Chem. Soc. 1991; 113: 6294Crossref Scopus (490) Google Scholar, 10.Katta V. Chait B.T. J. Am. Chem. Soc. 1991; 113: 8534-8535Crossref Scopus (431) Google Scholar, 11.Lee E.D. Mück W. Henion J.D. Covey T.R. J. Am. Chem. Soc. 1989; 111: 4600-4604Crossref Scopus (101) Google Scholar, 12.Hsieh F.Y.L. Tong X. Wachs T. Ganem B. Henion J. Anal. Biochem. 1995; 229: 20-25Crossref PubMed Scopus (53) Google Scholar, 13.Wu J. Takayama S. Wong C.H. Siuzdak G. Chem. Biol. (Lond.). 1997; 4: 653-657Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 14.Gerber S.A. Scott C.R. Turecek F. Gelb M.H. J. Am. Chem. Soc. 1999; 121: 1102-1103Crossref Scopus (44) Google Scholar, 15.Kriwacki R. Wu J. Tennant L. Wright P.E. Siuzdak G. J. Chromatogr. Sect. A. 1997; 777: 23-30Crossref PubMed Scopus (52) Google Scholar) especially for reactions that occurin vivo or are dynamic in nature. For example, the quantitative measurement of viral capsid proteolytic fragments offers a unique approach toward studying capsid dynamics (16.Bothner B. Dong X-F.A. Bibbs L.D. Johnson J.E. Siuzdak G. J. Biol. Chem. 1998; 273: 673-676Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17.Bothner B. Schneemann A. Marshall D. Reddy V. Johnson J.E. Siuzdak G. Nat. Struct. Biol. 1999; 6: 114-116Crossref PubMed Scopus (73) Google Scholar) (as of yet, no other spectroscopic methods exist to perform such mobility measurements). In addition, while spectroscopic probes exist for monitoring small molecule, enzyme-catalyzed reactions, these reactions typically require the release of a chromophore as the reaction proceeds. Unfortunately, the preparation of chromogenic substrates often requires substrate modification that can alter reaction kinetics or necessitate time consuming, multistep synthesis. Therefore, assay methodologies that allow for the rapid and quantitative measurement of enzyme activity, offer novel approaches toward monitoring protein interactions, and obviate the need for substrate modification (1.Matayoshi E.D. Wang G.T. Kraft G.A. Erickson J. Science. 1990; 247: 954-958Crossref PubMed Scopus (556) Google Scholar, 13.Wu J. Takayama S. Wong C.H. Siuzdak G. Chem. Biol. (Lond.). 1997; 4: 653-657Abstract Full Text PDF PubMed Scopus (58) Google Scholar) would be very beneficial. In recent years, mass spectrometry has emerged as a valuable tool in biochemistry offering unique insight into biological systems with respect to sensitivity and accuracy (2.Siuzdak G. Mass Spectrometry for Biotechnology. Academic Press, San Diego1996Google Scholar, 3.Hunt D.F. Michel H. Dickinson T.A. Shabanowitz J. Cox A.L. Sakaguchi K. Appella E. Grey H.M. Sette A. Science. 1992; 256: 1817-1820Crossref PubMed Scopus (631) Google Scholar, 4.Chait B.T. Wang R. Beavis R.C. Kent S.B. Science. 1993; 262: 89Crossref PubMed Scopus (268) Google Scholar, 5.Miranker A. Robinson C.V. Radford S.E. Dobson C.M. FASEB J. 1996; 10: 93Crossref PubMed Scopus (165) Google Scholar, 6.Kouach M. Belaiche D. Jaquinod M. Couppez M. Kmiecik D. Ricart G. Van Dorsselaer A. Sautiere P. Briand G. Biol. Mass Spectrom. 1994; 23: 283Crossref PubMed Scopus (16) Google Scholar, 7.Krebs J.F. Siuzdak G.H. Dyson J. Stewart J.D. Benkovic S.J. Biochemistry. 1995; 34: 720Crossref PubMed Scopus (36) Google Scholar, 8.Siuzdak G. Krebs J.F. Benkovic S.J. Dyson J.H. J. Am. Chem. Soc. 1994; 116: 7937Crossref Scopus (27) Google Scholar, 9.Ganem B. Li Y.T. Henion J.D. J. Am. Chem. Soc. 1991; 113: 6294Crossref Scopus (490) Google Scholar, 10.Katta V. Chait B.T. J. Am. Chem. Soc. 1991; 113: 8534-8535Crossref Scopus (431) Google Scholar, 11.Lee E.D. Mück W. Henion J.D. Covey T.R. J. Am. Chem. Soc. 1989; 111: 4600-4604Crossref Scopus (101) Google Scholar, 12.Hsieh F.Y.L. Tong X. Wachs T. Ganem B. Henion J. Anal. Biochem. 1995; 229: 20-25Crossref PubMed Scopus (53) Google Scholar, 13.Wu J. Takayama S. Wong C.H. Siuzdak G. Chem. Biol. (Lond.). 1997; 4: 653-657Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 14.Gerber S.A. Scott C.R. Turecek F. Gelb M.H. J. Am. Chem. Soc. 1999; 121: 1102-1103Crossref Scopus (44) Google Scholar). More specifically, mass spectrometry permits protein sequencing (3.Hunt D.F. Michel H. Dickinson T.A. Shabanowitz J. Cox A.L. Sakaguchi K. Appella E. Grey H.M. Sette A. Science. 1992; 256: 1817-1820Crossref PubMed Scopus (631) Google Scholar, 4.Chait B.T. Wang R. Beavis R.C. Kent S.B. Science. 1993; 262: 89Crossref PubMed Scopus (268) Google Scholar), the elucidation of protein folding pathways (5.Miranker A. Robinson C.V. Radford S.E. Dobson C.M. FASEB J. 1996; 10: 93Crossref PubMed Scopus (165) Google Scholar), for characterizing post-translational modifications on peptides and proteins (6.Kouach M. Belaiche D. Jaquinod M. Couppez M. Kmiecik D. Ricart G. Van Dorsselaer A. Sautiere P. Briand G. Biol. Mass Spectrom. 1994; 23: 283Crossref PubMed Scopus (16) Google Scholar), and for detecting covalent and noncovalent protein-ligand complexes (7.Krebs J.F. Siuzdak G.H. Dyson J. Stewart J.D. Benkovic S.J. Biochemistry. 1995; 34: 720Crossref PubMed Scopus (36) Google Scholar, 8.Siuzdak G. Krebs J.F. Benkovic S.J. Dyson J.H. J. Am. Chem. Soc. 1994; 116: 7937Crossref Scopus (27) Google Scholar, 9.Ganem B. Li Y.T. Henion J.D. J. Am. Chem. Soc. 1991; 113: 6294Crossref Scopus (490) Google Scholar, 10.Katta V. Chait B.T. J. Am. Chem. Soc. 1991; 113: 8534-8535Crossref Scopus (431) Google Scholar). Moreover, the accuracy and reproducibility of the technique make it well suited for the quantitative study of enzyme-catalyzed reactions (11.Lee E.D. Mück W. Henion J.D. Covey T.R. J. Am. Chem. Soc. 1989; 111: 4600-4604Crossref Scopus (101) Google Scholar, 12.Hsieh F.Y.L. Tong X. Wachs T. Ganem B. Henion J. Anal. Biochem. 1995; 229: 20-25Crossref PubMed Scopus (53) Google Scholar, 13.Wu J. Takayama S. Wong C.H. Siuzdak G. Chem. Biol. (Lond.). 1997; 4: 653-657Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 14.Gerber S.A. Scott C.R. Turecek F. Gelb M.H. J. Am. Chem. Soc. 1999; 121: 1102-1103Crossref Scopus (44) Google Scholar). Here the utility of electrospray ionization (ESI)1 and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is examined for the qualitative and quantitative characterization of viral capsid dynamics, as well as the hydrolysis of small molecules with α-glucosidase and bovine lipoprotein lipase. The measurement of proteolytic fragments, as well as k cat andK m provides quantitative insight into viral capsid dynamics, verifies UV results, demonstrates the utility of an internal standard for accurate kinetic measurements, and extends its utility to the difficult analysis of lipase-catalyzed hydrolysis of an unmodified substrate. All organic solvents were purchased from Aldrich. Bovine lipoprotein lipase was purchased from Sigma. 1,2-O-Dilauryl-rac-glycero-3-glutaric acid resorufin ester was purchased from Roche Molecular Biochemicals. Resorufin and glutaric acid were purchased from Aldrich. 1,2-O-Dilauryl-rac-glycerol, 1,2-O-dilauryl-rac-glycero-3-glutaric acid, 1,2-O-dilauryl-rac-glycero-3-benzyl ether, 1,2-O-dilauryl-rac-glycero-3-glutaric acid hexyl ester, and 1,2-O-dilauryl-rac-glycero-3-lauric acid ester were synthesized using the following method. 1,2-O-Glycerol acetonide was generated by refluxing a glycerol/acetone solution in the presence of p-toluene sulfonic acid. 1,2-O-Glycerol acetonide was then converted to the benzyl ether by deprotonation of the alcohol with sodium hydride in tetrahydrofuran followed by alkylation with benzyl bromide. The protected benzyl ether was subsequently deprotected with aqueous acid. The resulting glycerol-1-benzyl ether was deprotonated with 2.5 eq of sodium hydride and alkylated with 2.5 eq of lauryl bromide (1-bromododecane) at 0 °C. The reaction was allowed to stand for 24 h at 25 °C. After purification by flash chromatography, 1,2-O-dilauryl-rac-glycerol benzyl ether was obtained in 53% yield. The target compound, 1,2-O-dilauryl-rac-glycerol, was obtained by removal of the benzyl ether protection group by hydrogenation (H2/Pd/C, ethanol, 24 h). The resulting 1,2-O-dilauryl-rac-glycerol was obtained by flash chromatography in 95% yield. 1,2-O-Dilauryl-rac-glycerol was converted to 1,2-O-dilauryl-rac-glycero-3-glutaric acid by refluxing the diether with glutaric anhydride in the presence of 4-(dimethylamino)pyridine in pyridine solvent. After acidification and aqueous workup the target glutaric acid derivative was obtained in 72% yield. 1,2-O-Dilauryl-rac-glycero-3-glutaric acid hexyl ester was synthesized in 90% yield by the condensation of 1,2-O-dilauryl-rac-glycero-3-glutaric acid and 1-hexanol at room temperature with 1,3-dicyclohexylcarbodiimide in methylene chloride and 4-pyrrolidinopyridine as a catalyst. Lauric acid, CH3(CH2)10COOH, was first converted to the acid chloride by reaction with oxalyl chloride in benzene. The resulting acid chloride was added to a mixture containing 1,2-O-dilauryl-rac-glycerol, pyridine, and benzene at room temperature. 1,2-O-Dilauryl-rac-glycero-3-lauric acid ester was obtained in 96% yield. ESI experiments were performed using a Hewlett-Packard MSD, Perkin-Elmer SCIEX API 100 and API III mass spectrometers. LC/MS measurements were performed on a Hewlett-Packard 1090 MSD. MALDI-MS measurements were performed on a PerSeptive Voyager STR time-of-flight reflectron instrument. UV-visible measurements were performed on a Perkin-Elmer UV spectrophotometer attached to a constant temperature regulator. All experiments were conducted at 25 °C. 20 μg (2.5 pmol) of wild type FHV and synthetic FHV (sFHV) were prepared to a final concentration of 0.5 mg/ml in 25 mm Tris buffer at pH 7.5. 0.5 μl of the trypsin stock solution at 0.02 μg/μl was added to FHV to start the digestion and the reactions were terminated by the addition of 10 μl of acetic acid after 1.5 h. Limited trypsin digestion experiments were performed at room temperature at an E:S ratio of 1:2000 and the digestion was monitored as a function of time at different time points (5 min, 10 min, 15 min, 30 min, 1 h, and 1.5 h) using MALDI-MS. The LC/MS measurements were performed on the viruses after 1.5 h of digestion. Ethanol precipitation was used to remove viral RNA from tryptic peptides, which (if not removed) affected the quality of the LC separation. To perform ethanol precipitation, 0.5 μl of glycogen (2 mg/ml), 4.5 μl of 3 m NaOAc (pH 3.5), and 135 μl of ethanol was added to the digest. After centrifugation, the sample supernatants were transferred to separate Microfuge tubes, and dried under vacuum. The samples were then dissolved in H2O to a starting volume of 40 μl. Selected ion monitoring was used in LC/MS and a synthetic peptide was used as an internal standard. The synthetic peptide (VVTTTQTAPVPQQNVPR) was synthesized for its similarity to the sequence and ionization properties of the selected viral peptide (from β-protein 15–32: VVVTTTQTAPVPQQNVPR). A Supelcosil ABZ+ (3.3 cm × 2.1 mm, 3 mm) column was used to achieve rapid and high resolution separation. 10 μl of digest samples were injected for every LC/MS analysis. Solvent A was 0.03% trifluoroacetic acid in H2O and solvent B was 0.03% trifluoroacetic acid in ACN/H2O (95/5, v/v) and the chromatography (5–25% B gradient) was performed over 8 min. The LC/MS chromatography experiments were performed with an HP1090 liquid chromatograph and an HP1100 electrospray mass selective detector. The kinetics of α-glucosidase (brewers' yeast) were determined by measuring the concentration ofp-nitrophenylate by ESI-MS and UV-visible spectroscopy in the presence of an internal standard. A calibration curve was generated starting with 2 μm p-nitrophenyl-α-glucopyranoside, 2 μm p-nitrophenol, and 2 μmglucose, all in 25 μm sodium phosphate buffer (pH 7.5), and 2 μm 8-anilino-1-naphthylsulfonic acid inN,N-dimethylformamide, as internal standard, for experiments in negative mode. For end point kinetic studies, synthetic mixtures of the above reagents were prepared for calibration purposes by dilution. Substrate (1) concentration ranged from 0 to 120 μm, and product (2 and 3) concentration ranged from 0 to 120 μm, while the internal standard concentration constant was kept at 40 μm. Reactions were initiated by adding 10 μl of α-glucosidase (34 nm in 25 mm sodium phosphate, pH 7.5) to 990 μl of the reaction mixture, and the kinetics followed both by UV-visible at 452 nm and ESI-MS. For ESI-MS, reaction samples were stopped by taking 20-μl aliquots and diluting with 20 μl of methanol. Samples were immediately injected into the ESI-MS. Allk cat and K m experiments were done in negative mode using 2 μm8-anilino-1-naphthylsulfonic acid in N,N-dimethylformamide, as internal standard. The kinetics of bovine lipoprotein lipase were monitored with ESI-MS by determining the response of the sodium adducts of 1,2-O-dilauryl-rac-glycerol 5, and 1,2-O-dilauryl-rac-glycero-3-glutaric acid6, in the presence of 1,2-O-dilauryl-rac-glycero-3-benzyl ether, as the internal standard. 2 μm Dioxane solutions of 1,2-O-dilauryl-rac-glycerol 5,1,2-O-dilauryl-rac-glycero-3-glutaric acid6,1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester 4, and 1,2-O-dilauryl-rac-glycero-3-benzyl ether were used for calibration purposes. Synthetic mixtures of the above reagents were used to prepare two separate calibration curves by dilution, so that in one case the concentration of substrate 4 was varied from 0 to 120 μm, and the concentration of the alcohol product5 was varied from 0 to 120 μm, while keeping the concentration of the acid 6 and the internal standard constant at 40 μm; in the other case, the concentration of substrate 4 was varied from 0 to 120 μm, and the concentration of the acid product 6 was varied from 0 to 120 μm, while keeping the concentration of the alcohol 5 and the internal standard constant at 40 μm. Reactions were initiated by adding 10 μl of bovine lipoprotein lipase (0.2 nm in 25 mm sodium phosphate, pH 7.0) to 990 μl of the reaction mixture, and the kinetics followed both by ESI-MS and UV-visible spectroscopy at 592 nm. For ESI-MS, reaction samples (20 μl aliquots) were removed periodically and stopped by diluting with 200 μl of dioxane. Samples were then immediately frozen until analyzed by ESI-MS in positive mode. The combined use of enzymology with mass spectrometry is providing new insight into the quantitative aspects of enzyme-catalyzed reactions such as glycosidation and ester hydrolysis, as well as in protein structure determination (15.Kriwacki R. Wu J. Tennant L. Wright P.E. Siuzdak G. J. Chromatogr. Sect. A. 1997; 777: 23-30Crossref PubMed Scopus (52) Google Scholar). In these experiments viral capsid proteins were examined using limited proteolysis combined with MALDI-MS (16.Bothner B. Dong X-F.A. Bibbs L.D. Johnson J.E. Siuzdak G. J. Biol. Chem. 1998; 273: 673-676Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17.Bothner B. Schneemann A. Marshall D. Reddy V. Johnson J.E. Siuzdak G. Nat. Struct. Biol. 1999; 6: 114-116Crossref PubMed Scopus (73) Google Scholar, 18.Siuzdak G. J. Mass Spectrom. 1998; 33: 203-211Crossref PubMed Scopus (60) Google Scholar), and ESI-MS was used as a means of generating quantitative information on protein capsid mobility. In previous experiments, enzyme digestion was used to map the surface proteins on the virus, leading to the fascinating evidence that the capsid proteins are mobile (16.Bothner B. Dong X-F.A. Bibbs L.D. Johnson J.E. Siuzdak G. J. Biol. Chem. 1998; 273: 673-676Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17.Bothner B. Schneemann A. Marshall D. Reddy V. Johnson J.E. Siuzdak G. Nat. Struct. Biol. 1999; 6: 114-116Crossref PubMed Scopus (73) Google Scholar, 18.Siuzdak G. J. Mass Spectrom. 1998; 33: 203-211Crossref PubMed Scopus (60) Google Scholar). Recently, trypsin digestion between active whole virus and isolated capsid proteins were compared using MALDI-MS, where the isolated capsid proteins were found to be significantly more susceptible to proteolysis than proteins in the intact capsid (Fig.1). This result was consistent with the intact virus being highly protected both internally (by RNA) and externally (by other viral capsid proteins). In addition, the exact sites of proteolysis on the intact virus provided interesting insight into particle-structure dynamics. Examination of the tryptic digestion fragments from the intact flock house virus (FHV, composed of 180 copies of the β-protein and γ-peptide, containing 363 and 44 amino acids, respectively) revealed that the first sites to be cleaved were, based on crystallographic data, located internal to the viral capsid. Based on the known sequence of the capsid proteins it was possible to map out the digestion course on the FHV capsid for the major β-protein. At low enzyme-to-substrate ratios, time-resolved digestion of the intact virus was observed to start at the N terminus of β-protein and moved inward. Sequentially, trypsin cleaved the β-protein at positions 10, 13, 31, and 34 in the first 15 min, reaching residue 57 after 3 h of incubation. Additional experiments performed with clostripain (Arg-C) also showed that digestion initially occurred on the N-terminal of the β-protein in the intact virus. Since the β-proteins N terminus is internal to the viral capsid (based on the crystal structure), the observed cleavage are believed to reflect transient exposure of these regions to the viral surface (16.Bothner B. Dong X-F.A. Bibbs L.D. Johnson J.E. Siuzdak G. J. Biol. Chem. 1998; 273: 673-676Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17.Bothner B. Schneemann A. Marshall D. Reddy V. Johnson J.E. Siuzdak G. Nat. Struct. Biol. 1999; 6: 114-116Crossref PubMed Scopus (73) Google Scholar, 18.Siuzdak G. J. Mass Spectrom. 1998; 33: 203-211Crossref PubMed Scopus (60) Google Scholar). Trypsin proteolysis experiments performed on a mutant version of FHV (deleted at N terminus residues 2–31 of the β-protein) also produced initial sites of cleavage via sequential digestion from the truncated N terminus albeit at a significantly slower rate than observed for wild type FHV. These results indicate that in both the wild type and mutant versions of FHV a significant portion of the N terminus is being transiently exposed on the surface of the intact virus. Further suggesting that, in solution, the intact virus undergoes significant conformational changes. To further elucidate the relationship between viral dynamics and intermolecular interactions within the virus, a virus-like particle (sFHV) was examined with respect to the authentic wild type virus (FHV). The synthetic virus has the same capsid protein as wild type FHV, yet the particle contains random cellular RNA instead of authentic viral RNA. The difference between the two viral systems is that significantly less RNA-capsid interaction exists in sFHV than FHV (17.Bothner B. Schneemann A. Marshall D. Reddy V. Johnson J.E. Siuzdak G. Nat. Struct. Biol. 1999; 6: 114-116Crossref PubMed Scopus (73) Google Scholar), a difference that should be observed in viral capsid mobility. Relative capsid dynamics were studied by trypsin digestion experiments performed in parallel (and reproduced in five separate experiments), where the rate of proteolysis was determined by LC/MS using a synthetic version of the capsid β-protein(16–32) as an internal standard. A representative separation is shown in Fig.2 where relative ion intensities of the released peptide fragments were used to allow comparisons between reactions. Consistent with previous experiments (17.Bothner B. Schneemann A. Marshall D. Reddy V. Johnson J.E. Siuzdak G. Nat. Struct. Biol. 1999; 6: 114-116Crossref PubMed Scopus (73) Google Scholar), a dramatic increase in proteolysis was seen in sFHV, thereby supporting the idea that decreased intermolecular reactions decrease the stability of the virus. sFHV is more susceptible to trypsin digestion, which has a digestion rate six times higher than FHV, indicating a large stabilizing effect due to the authentic RNA. Because of the inherent accuracy of electrospray ionization measurements, especially when used in conjunction with an internal standard, these quantitative measurements present a solution-phase representation of the change in dynamics that correspond to the presence of the authentic viral RNA. Reactions such as lipid ester hydrolysis and oligosaccharide glycosidation involve substrates and products that are readily analyzed and quantified by mass spectrometry (13.Wu J. Takayama S. Wong C.H. Siuzdak G. Chem. Biol. (Lond.). 1997; 4: 653-657Abstract Full Text PDF PubMed Scopus (58) Google Scholar), which is offering a new window into these enzyme-catalyzed reactions. α-Glucosidase catalyzes the hydrolysis of linear oligosaccharides and releases one glucose residue at a time. This enzyme has been extensively characterized, and a variety of substrates are available that permit direct comparison of the mass spectrometry data with independent kinetic measurements. For this study, p-nitrophenyl-α-glucopyranoside (1) was chosen as a substrate which can be cleaved by α-glucosidase, SchemeFS1, to produce thep-nitrophenolate ion (2) and glucose (3). Selected ion monitoring was used to generate a calibration curve for reaction products 2 and 3(in the concentration range 0–1 mm) with either 8-anilino-1-naphthalenesulfonic acid (negative mode) or methyltriphenyl phosphonium ion (positive mode) as internal standards. The sodium ion adducts provided the best response and an excellent correlation between concentration and selected ion peak intensity were observed (r 2 = 0.998 for glucose, and r = 0.994 for p-nitrophenolate). The hydrolysis of 1 (10 μm to 1 mm) was performed at 25 °C in 25 mm sodium phosphate buffer (pH 7.5) containing either 8-anilino-1-naphthylsulfonic acid (40 μm) or methyltriphenylphosphonium bromide (2.5–500 μm) in the presence of α-glucosidase from brewers' yeast (Sigma). Aliquots were periodically removed from the reaction, diluted with methanol (1:2), and immediately analyzed by ESI-MS. Typical ESI-MS results are shown in Fig. 3. The change in intensity of the product ion relative to the internal standard was determined as a function of time. Product concentrations were then calculated directly from the calibration curve. Fig. 3 shows the reaction progress for the hydrolysis of 1, monitored by the increase of thep-nitrophenolate ion concentration UV-visible at 400 nm and by ESI-MS. At a concentration of 120 μm 1, the initial rates observed by UV-visible spectroscopy and ESI-MS were 4.82 and 4.92 μm/min, respectively. Similar quality data was obtained when the rate of formation of glucose was monitored by ESI-MS and compared with the rate of formation of p-nitrophenolate, as determined by UV-visible spectroscopy. Compound 1 at a concentration of 1.0 mm generated initial rates of 11.7 and 11.5 μm/min, observed by UV-visible spectroscopy at 452 nm and ESI-MS, respectively. Both experiments illustrate the good quality of data that can be obtained over the time course of the reaction. Fig. 4 shows a plot generated from the ESI-MS data where, in order to determine k catand K m, the initial rates of hydrolysis in Scheme FS1were obtained. The kinetic parameters calculated arek cat = 256 ± 27 min−1 andK m = 0.535 ± 0.192 μm. For comparison, the k cat and K mvalues of 266 ± 30 min−1 and 0.407 ± 0.077 μm were determined by UV-visible spectroscopy, showing good agreement between the two methods. This approach was compared with the original work by Henion et al. (11.Lee E.D. Mück W. Henion J.D. Covey T.R. J. Am. Chem. Soc. 1989; 111: 4600-4604Crossref Scopus (101) Google Scholar, 12.Hsieh F.Y.L. Tong X. Wachs T. Ganem B. Henion J. Anal. Biochem. 1995; 229: 20-25Crossref PubMed Scopus (53) Google Scholar), whose investigations initiated the use of electrospray mass spectrometry for monitoring enzyme reactions. In addition, it was found (by direct comparison to UV-visible measurements) that addition of an internal standard further increased the accuracy of the ESI-MS results. Performing kinetic measurements of lipases is difficult because of the limited solubility of the substrates can result in precipitation and aggregation (micelle formation). With this in mind, lipase-catalyzed hydrolysis of triacylglycerides and related substrates has been studied (19.Wang C. Hartsuck J. Downs D. Bass H. Biochim. Biophys. Acta. 1990; 1043: 143Crossref PubMed Scopus (5) Google Scholar, 20.Quinn D. Shirai K. Jackson R. Harmony J. Biochemistry. 1982; 21: 6872Crossref PubMed Scopus (74) Google Scholar, 21.Jackson, R. (1983) The Enzymes, Vol. XVI, Chapter 5, Academic Press, New YorkGoogle Scholar), where a variety of available substrates have been used to allow for a direct comparison of the mass spectrometry data with independent kinetic determinations. For example, 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester (4) has been reported to be an excellent substrate for lipases. Its hydrolysis has been described as a series of steps involving first the lipase-catalyzed formation of 1,2-O-dilaurylglycerol and glutaric acid-resorufin ester, followed by the spontaneous intramolecularly assisted hydrolysis of the latter product to release resorufin, as shown in SchemeFS2 , path a (22.Chemnitius G. Erdmann H. Schmid R. Anal. Biochem. 1992; 202: 16Crossref PubMed Scopus (11) Google Scholar, 23.Neumann, U., Junius, M., and Batz, H. G. (1989) U. S. Patent 4847376Google Scholar,26.Neumann, U., Junius, M., and Batz, H. G. (1991) U. S. Patent 4988497Google Scholar). This proposed mechanism is based on absorbance data and the fact that intramolecularly assisted chromophore release has been reported in other investigations (24.Washburn W.N. Dennis E.A. J. Am. Chem. Soc. 1990; 112: 2040Crossref Scopus (21) Google Scholar, 25.Gong B. Lesley S.A. Schultz P.G. J. Am. Chem. Soc. 1992; 114: 1486Crossref Scopus (17) Google Scholar). Indeed, it was confirmed that the resorufin substrate is processed rapidly by bovine lipoprotein lipase. The time course of the reaction is monitored readily by the release of the resorufin chromophore at 572 nm. The initial rate was 39.7 μm/min/mg of enzyme at 25 °C when the substrate concentration was 120 μm in 25 mm sodium phosphate buffer (pH 7.0), containing 20% dioxane and acetonitrile. To monitor the lipase reaction by ESI-MS (Fig.5), selected ion monitoring was used first to generate a calibration curve for the alcohol product, 1,2-O-dilauryl-rac-glycerol (6), using 1,2-O-dilauryl-rac-glycero-3-benzyl ether as an internal standard. Aliquots of the lipase reaction were taken at various time intervals, diluted with dioxane, and then frozen until analyzed by ESI. Surprisingly, little of the expected product5 was obtained. Instead of the glycerol ester of substrate4 being cleaved, it was found that the resorufin ester was initially cleaved (Scheme FS2, path b). As shown in Scheme FS2, initial formation of the acid ester product6 correlates directly with the release of the resorufin chromophore. The initial rate of formation of the acid ester is 33.6 μm/min/mg, in agreement with the value obtained spectrophotometrically. At longer times the concentrations of the acid ester 6, as determined by ESI-MS, differ from those determined spectrophotometrically, possibly due to some precipitation of the product during the reaction and after the freezing procedure. The conversion of 6 to the alcohol 5 is very slow (0.186 μm/min/mg), where the initial rate of formation of5 from 4 is 3.19 μm/min/mg. Contrary to expectations, the large aromatic resorufin-leaving group may orient the substrate within the active site in such a way as to favor cleavage of the activated ester over cleavage of the glycerol ester. The unique capabilities of ESI-MS thus provide direct insight into molecular changes that take place. Because the activated diester 4 partitioned in a manner that was unexpected, the partitioning of a non-activated dialkyl ester, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-hexyl ester 7, by lipoprotein lipase was examined to determine the extent to which the chromophore alters the course of the reaction (Scheme FS3). This substrate lacks a chromophore, making its analysis difficult by other techniques. The formation of both alcohol 5 and acid ester 6 ions was monitored, and it was found that cleavage of the glycerol ester (path a, Scheme FS3) is preferred over the terminal alkyl ester by a factor of 6.7:1. The initial rate of 5 is 46.1 μm/min/mg, approximately the same as that of the resorufin substrate, which suggests that the resorufin chromophore greatly perturbs the normal processing of the glycerol esters. The hydrolysis of a more “natural” ester was also examined to place the above results in their proper context. The kinetics of 1,2-O-dilauryl-rac-glycero-3-lauric acid ester8 showed a clean conversion of ester to product 5and 9 in the presence of lipoprotein lipase (SchemeFS4). The initial rate of cleavage increased to 109 μm/min/mg.Figure FS4Hydrolysis of non-activated dialkyl ester, 1,2-O-dilauryl-rac-glycero-3-lauric acid ester (8.Siuzdak G. Krebs J.F. Benkovic S.J. Dyson J.H. J. Am. Chem. Soc. 1994; 116: 7937Crossref Scopus (27) Google Scholar) catalyzed by lipoprotein lipase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These experiments illustrate that mass spectrometry is qualitatively useful for examining reaction pathways and protein structure, and is also a viable approach for quantitative determination of kinetics. From macromolecular protein dynamics, kinetic measurements, and the effect of unnatural substrates on reaction dynamics, mass spectrometry offers excellent accuracy, reproducibility, and is especially well suited for assaying reactions that cannot be followed spectrophotometrically. In cases where introduction of a chromophore drastically changes the fate of the reaction as a result of the structural features of the substrate (as illustrated by the resorufin-containing substrate), ESI-MS has been found to be especially valuable. The small sample size, minimal handling requirements, along with the potential for high throughput represent further significant advantages. Accurate mass measurements by ESI-MS also suggest the further utility of this technique for the kinetic analysis of reactions involving enzyme inhibitors (13.Wu J. Takayama S. Wong C.H. Siuzdak G. Chem. Biol. (Lond.). 1997; 4: 653-657Abstract Full Text PDF PubMed Scopus (58) Google Scholar)." @default.
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