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Determination of Disulfide Bond Assignment of Human Vitamin K-dependent γ-Glutamyl Carboxylase by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês

10.1074/jbc.m309164200

1083-351X

Jian‐Ke Tie, Vasantha P. Mutucumarana, David L. Straight, Kevin Carrick, R. Marshall Pope, Darrel W. Stafford,

Alcoholism and Thiamine Deficiency

Vitamin K-dependent γ-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to γ-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al. in Pudota, B. N., Miyagi, M., Hallgren, K. W., West, K. A., Crabb, J. W., Misono, K. S., and Berkner, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13033–13038 reported that Cys-99 and Cys-450 are the carboxylase active site residues. We determined the form of all cysteines in carboxylase using in-gel protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92–100 (Cys-99) disulfide-linked with peptide 446–453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase. Vitamin K-dependent γ-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to γ-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al. in Pudota, B. N., Miyagi, M., Hallgren, K. W., West, K. A., Crabb, J. W., Misono, K. S., and Berkner, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13033–13038 reported that Cys-99 and Cys-450 are the carboxylase active site residues. We determined the form of all cysteines in carboxylase using in-gel protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92–100 (Cys-99) disulfide-linked with peptide 446–453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase. The vitamin K-dependent carboxylase is an integral membrane glycoprotein that catalyzes the post-translational modification of specific glutamic acid residues to γ-carboxyglutamic acid (Gla) 1The abbreviations used are: Gla, γ-carboxyglutamic acid; ER, endoplasmic reticulum; PRGP, proline-rich γ-carboxyglutamic acid proteins; TMG, transmembrane γ-carboxyglutamic acid proteins; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; NEM, N-ethylmaleimide; TCEP, tris(2-carboxyethyl)phosphine; MOPS, 3-(N-morpholino)propanesulfonic acid; CID, collisionally induced dissociation; LC-ESMS, liquid chromatography electrospray mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; ESI-MS, electrospray ionization mass spectrometry. (1Presnell S.R. Stafford D.W. Thromb. Haemost. 2002; 87: 937-946Crossref PubMed Scopus (82) Google Scholar, 2Price P.A. Annu. Rev. Nutr. 1988; 8: 565-583Crossref PubMed Scopus (150) Google Scholar). The carboxylation reaction occurs in the lumen of the ER (3Bristol J.A. Ratcliffe J.V. Roth D.A. Jacobs M.A. Furie B.C. Furie B. Blood. 1996; 88: 2585-2593Crossref PubMed Google Scholar, 4Carlisle T.L. Suttie J.W. Biochemistry. 1980; 19: 1161-1167Crossref PubMed Scopus (73) Google Scholar) and uses the substrates carbon dioxide, oxygen, and vitamin K hydroquinone. During the process of carboxylation, the γ-proton of the glutamic acid is abstracted, followed by the addition of carbon dioxide (5Vermeer C. Biochem. J. 1990; 266: 625-636Crossref PubMed Scopus (283) Google Scholar). Simultaneous with carboxylation, the vitamin K hydroquinone is converted to vitamin K epoxide, which is converted back to vitamin K by the enzyme epoxide reductase. The formation of vitamin K epoxide has sometimes been called an epoxidation reaction. Gla modification is critical for the function of more than a dozen proteins involved in blood coagulation and calcium homeostasis (6Furie B. Bouchard B.A. Furie B.C. Blood. 1999; 93: 1798-1808Crossref PubMed Google Scholar, 7Berkner K.L. J. Nutr. 2000; 130: 1877-1880Crossref PubMed Scopus (56) Google Scholar). The importance of vitamin K-dependent proteins may be even greater than previously thought, as evidenced by the discovery of growth-arrest protein gas-6 (8Manfioletti G. Brancolini C. Avanzi G. Schneider C. Mol. Cell. Biol. 1993; 13: 4976-4985Crossref PubMed Scopus (532) Google Scholar), and the very recent identification of four putative vitamin K-dependent membrane Gla proteins PRGP1, PRGP2, TMG3, and TMG4 (9Kulman J.D. Harris J.E. Haldeman B.A. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9058-9062Crossref PubMed Scopus (94) Google Scholar, 10Kulman J.D. Harris J.E. Xie L. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1370-1375Crossref PubMed Scopus (91) Google Scholar). There are ten cysteine residues in the human carboxylase molecule. Our work on the topology of the carboxylase predicts that of these ten cysteines, two are located in the cytoplasm, three are buried in the ER membrane, and five are found in the lumen of the ER (11Tie J. Wu S.M. Jin D. Nicchitta C.V. Stafford D.W. Blood. 2000; 96: 973-978Crossref PubMed Google Scholar). Sulfhydryl groups and disulfide bonds are important for both the structure and function of proteins (12Giles N.M. Giles G.I. Jacob C. Biochem. Biophys. Res. Commun. 2003; 300: 1-4Crossref PubMed Scopus (165) Google Scholar, 13Hiniker A. Bardwell J.C. Biochemistry. 2003; 42: 1179-1185Crossref PubMed Scopus (30) Google Scholar, 14Wedemeyer W.J. Welker E. Narayan M. Scheraga H.A. Biochemistry. 2000; 39: 4207-4216Crossref PubMed Scopus (525) Google Scholar, 15Molinari M. Helenius A. Nature. 1999; 402: 90-93Crossref PubMed Scopus (274) Google Scholar). For example, the natural abundance of cysteine is 1.2%, but these residues constitute 5.6% of enzyme catalytic sites (16Bartlett G.J. Porter C.T. Borkakoti N. Thornton J.M. J. Mol. Biol. 2002; 324: 105-121Crossref PubMed Scopus (465) Google Scholar). Therefore, identification of free cysteine residues or those involved in disulfide bond formation can give valuable information about the structure and function of proteins. Several studies have implicated cysteine in the function of carboxylase. Chemical modification of carboxylase by sulfhydryl-reactive reagents suggests that cysteine residues are important for the carboxylation reaction (17Morris D.P. Soute B.A. Vermeer C. Stafford D.W. J. Biol. Chem. 1993; 268: 8735-8742Abstract Full Text PDF PubMed Google Scholar, 18Canfield L.M. Biochem. Biophys. Res. Commun. 1987; 148: 184-191Crossref PubMed Scopus (19) Google Scholar, 19Bouchard B.A. Furie B. Furie B.C. Biochemistry. 1999; 38: 9517-9523Crossref PubMed Scopus (19) Google Scholar, 20Canfield L.M. Sinsky T.A. Suttie J.W. Arch Biochem. Biophys. 1980; 202: 515-524Crossref PubMed Scopus (25) Google Scholar, 21Pudota B.N. Miyagi M. Hallgren K.W. West K.A. Crabb J.W. Misono K.S. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13033-13038Crossref PubMed Scopus (35) Google Scholar). Based on a non-enzymatic chemical model, Paul Dowd et al. (22Dowd P. Hershline R. Ham S.W. Naganathan S. Science. 1995; 269: 1684-1691Crossref PubMed Scopus (112) Google Scholar) developed a "base strength amplification mechanism" for carboxylation. They proposed that two free cysteines are involved in the active site of carboxylase. Recently, Pudota et al. (21Pudota B.N. Miyagi M. Hallgren K.W. West K.A. Crabb J.W. Misono K.S. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13033-13038Crossref PubMed Scopus (35) Google Scholar) analyzed the catalytically important cysteine residues of the carboxylase by modifying free cysteines with the radiolabeled sulfhydryl-reactive reagent 14C-NEM. These authors reported that cysteine residues 99 and 450 are the active site residues of carboxylase (21Pudota B.N. Miyagi M. Hallgren K.W. West K.A. Crabb J.W. Misono K.S. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13033-13038Crossref PubMed Scopus (35) Google Scholar). In contrast to the multiple studies on the importance of free cysteines in carboxylase, the only information about disulfide bridges in the structure of the carboxylase is the study by Wu et al. (23Wu S.M. Mutucumarana V.P. Geromanos S. Stafford D.W. J. Biol. Chem. 1997; 272: 11718-11722Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This study demonstrated that limited trypsin digestion of carboxylase at residues 349 and 351 results in a 30 kDa amino-terminal fragment disulfide linked to a 60 kDa carboxyl-terminal fragment. Although mass spectrometry has been used to characterize soluble proteins, its application to integral membrane proteins has lagged because of their unique characteristics (24Santoni V. Molloy M. Rabilloud T. Electrophoresis. 2000; 21: 1054-1070Crossref PubMed Scopus (831) Google Scholar, 25Wu C.C. Yates J.R. Nat. Biotechnol. 2003; 21: 262-267Crossref PubMed Scopus (506) Google Scholar). Integral membrane proteins represent around 30% of all proteins and play important roles in various cellular processes including signal transduction, cell adhesion, ion transport, endocytosis, and many enzymatic reactions (26Stevens T.J. Arkin I.T. Proteins. 2000; 39: 417-420Crossref PubMed Scopus (214) Google Scholar, 27Wallin E. von Heijne G. Protein Sci. 1998; 7: 1029-1038Crossref PubMed Scopus (1246) Google Scholar). The hydrophobic nature of the integral membrane proteins requires the use of detergents for solubilization and stability (28Garavito R.M. Ferguson-Miller S. J. Biol. Chem. 2001; 276: 32403-32406Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar), but most detergents are not compatible with mass spectrometric analysis. Therefore, new approaches for the application of mass spectrometry to characterize and identify membrane proteins are being actively pursued (25Wu C.C. Yates J.R. Nat. Biotechnol. 2003; 21: 262-267Crossref PubMed Scopus (506) Google Scholar, 29Wu C.C. MacCoss M.J. Howell K.E. Yates J.R. Nat. Biotechnol. 2003; 21: 532-538Crossref PubMed Scopus (606) Google Scholar, 30van Montfort B.A. Canas B. Duurkens R. Godovac-Zimmermann J. Robillard G.T. J. Mass Spectr. 2002; 37: 322-330Crossref PubMed Scopus (80) Google Scholar). Among recent studies, van Montfort et al. (30van Montfort B.A. Canas B. Duurkens R. Godovac-Zimmermann J. Robillard G.T. J. Mass Spectr. 2002; 37: 322-330Crossref PubMed Scopus (80) Google Scholar) reported an improved in-gel approach to generate peptide maps of integral membrane proteins for MALDI-MS. In this study, we have used in-gel protease digestion and MALDI-TOF MS/MS to identify the disulfide linkage of the human vitamin K-dependent carboxylase. Our results indicate that a disulfide bond joins cysteine residues 99 and 450. Mutation of either of these two cysteines significantly decreases the carboxylase activity. This is expected if the structure of the enzyme is compromised by the lack of the disulfide bond. Materials—All chemicals were reagent grade. CHAPS, n-octyl-β-d-glucopyranoside, α-cyano-4-hydroxysuccinnamic acid, and N-ethylmaleimide (NEM) were obtained from Sigma (St. Louis, MO). 1,2-Dioleoylsn-Glycero-3-Phosphocholine (PC) was from Avanti (Alabaster, AL). H-d-Phe-Pro-Arg-chloromethylketone and FLEEL were from Bachem (King of Prussia, PA). NaH14CO3 (specific activity, 54 mCi/mmol) was from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Tris-(2-carboxyethyl)phosphine (TCEP) hydrochloride was from Molecular Probes (Eugene, OR). Vitamin K1 was from Abbott Laboratories (Chicago, IL). PNGase F (500,000 units/ml) and all the restriction enzymes were from New England Biolabs (Beverly, MA). Aprotinin and sequencing grade modified trypsin and chymotrypsin were from Roche (Indianapolis, IN). The BacVector-3000 kit for insect cell transfection was from Novagen (Madison, WI). Coomassie Blue R-250, protein standard markers, and SDS-PAGE ready gel were from Bio-Rad. SP-Sepharose was from Amersham Biosciences, and anti-HPC4 resin was kindly provided by Dr. Charles Esmon (Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK). Site-directed Mutagenesis—Oligonucleotides and PCR primers used for site-directed mutagenesis were synthesized by Invitrogen Life Technologies (Frederick, MD). Site-directed cysteine mutagenesis of human carboxylase was conducted by the "Megaprimer" method of PCR mutagenesis (31Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar, 32Datta A.K. Nucleic Acids Res. 1995; 23: 4530-4531Crossref PubMed Scopus (100) Google Scholar). Wild-type human carboxylase cDNA with a FLAG tag (DYKDDDDK) at the amino terminus and a HPC4 tag (EDQVDPRLIDGK) at the carboxyl terminus (33Stearns D.J. Kurosawa S. Sims P.J. Esmon N.L. Esmon C.T. J. Biol. Chem. 1988; 263: 826-832Abstract Full Text PDF PubMed Google Scholar) was used as template DNA for the PCR. Mutations were screened by restriction digestion, verified by sequencing the entire cDNA of the carboxylase and subcloned to the expression vector pVL1392. Expression and Purification of Human Carboxylase in Insect Cells— Wild-type or cysteine mutant carboxylase cDNA engineered in the expression vector was co-transfected with BacVector 3000 triple-cut virus DNA into Sf9 cells. The recombinant virus was isolated by plaque purification, amplified, and screened by carboxylase activity assay of the cell lysate. Expression of carboxylase was done by infection of ∼2 × 106/ml High Five cells with the recombinant virus at a mutiplicity of infection of ∼1. Cells were collected after 48 h of infection, and the expressed carboxylase was purified by affinity chromatography using anti-HPC4 antibody-coupled Sepharose resin as described (34Stanley T.B. Jin D.Y. Lin P.J. Stafford D.W. J. Biol. Chem. 1999; 274: 16940-16944Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Further concentration of the protein was accomplished by binding the affinity-purified enzyme to SP-Sepharose in 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1% PC, 0.3% CHAPS, and 15% glycerol, followed by step elution with 500 mm NaCl in the same buffer. The reducing reagent dithiothreitol was excluded from all purification steps. Because our results on the activity of carboxylase with point mutations C343S and C288S differ from those of Pudota et al. (21Pudota B.N. Miyagi M. Hallgren K.W. West K.A. Crabb J.W. Misono K.S. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13033-13038Crossref PubMed Scopus (35) Google Scholar, 35Pudota B.N. Hommema E.L. Hallgren K.W. McNally B.A. Lee S. Berkner K.L. J. Biol. Chem. 2001; 276: 46878-46886Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), we isolated the virus DNA from which these mutant proteins were prepared and the entire cDNA sequence was again confirmed. Carboxylase Activity Assays—The carboxylase activity assay was performed in a total volume of 125 μl containing 25 mm MOPS, pH 7.5, 500 mm NaCl, 0.8 m (NH4)2SO4, 0.12% PC, 0.28% CHAPS, 4 μm proFIX, 5 μCi of NaH14CO3 (specific activity, 54 mCi/mmol), 222 μm vitamin K hydroquinone, and 1.25 mm FLEEL. The reaction was started by the addition of 10 μl of an ice-cold mix of NaH14CO3 and vitamin K hydroquinone; the reaction mix was immediately transferred to a 20 °C water bath and incubated for 30 min. The reactions were terminated by the addition of 1 ml of 5% trichloroacetic acid, and the amount of 14CO2 incorporated into the small substrate FLEEL was determined, as previously described (34Stanley T.B. Jin D.Y. Lin P.J. Stafford D.W. J. Biol. Chem. 1999; 274: 16940-16944Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Deglycosylation and NEM Modification of the Carboxylase—Glycoproteins often yield poor peptide maps for mass analysis because the oligosaccharides can effectively shield proteolytic cleavage sites (36Rudd P.M. Joao H.C. Coghill E. Fiten P. Saunders M.R. Opdenakker G. Dwek R.A. Biochemistry. 1994; 33: 17-22Crossref PubMed Scopus (284) Google Scholar). Therefore, freshly purified carboxylase was deglycosylated before being subjected to protease digestion by adding 100 μl of Buffer G7 (50 mm sodium phosphate, pH 7.5 at 25 °C) and 10 μl of PNGase F (500 units/μl) to a 900-μl carboxylase sample. Three hours at 37 °C was sufficient for deglycosylation of denatured carboxylase; however, 16 h at 18 °C was required for deglycosylation of native carboxylase. The efficiency of deglycosylation was evaluated by SDS-PAGE. NEM modification of carboxylase was performed at room temperature, as described by Pudota et al. (21Pudota B.N. Miyagi M. Hallgren K.W. West K.A. Crabb J.W. Misono K.S. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13033-13038Crossref PubMed Scopus (35) Google Scholar), except, instead of quenching the reaction by dithiothreitol, it was loaded directly onto non-reducing SDS-PAGE and the NEM used in our experiments was not radioactively labeled. In-gel Protease Digestion—Carboxylase samples were loaded onto 10% SDS-PAGE gels in the absence of reducing reagent. After separation, the gel was fixed with 25% isopropyl alcohol/10% acetic acid for 20 min and stained with 0.01% Coomassie Brilliant Blue R250 in 10% acetic acid for 1 h. It was destained with 10% acetic acid, the protein band excised, and the gel pieces treated as described (30van Montfort B.A. Canas B. Duurkens R. Godovac-Zimmermann J. Robillard G.T. J. Mass Spectr. 2002; 37: 322-330Crossref PubMed Scopus (80) Google Scholar). The gel pieces were completely destained with 50 mm NH4HCO3 in 40% ethanol, and then washed with 1000 μl of 25 mm NH4HCO3 three times for 15 min and cut into pieces of <1 mm3. Subsequently, the gel pieces were dehydrated with 1000 μl of acetonitrile three times for 10 min and completely dried with a SpeedVac. Protease digestion was started by the addition of sufficient 100 ng/μl sequencing grade-modified trypsin or chymotrypsin in 25 mm NH4HCO3 buffer to immerse the dried gel pieces. After re-hydration, the gel pieces were covered with an overlay of 20 μl of 25 mm NH4HCO3 buffer so that they remained immersed throughout the digestion. The protein was digested overnight by trypsin at 30 °C without agitation. Digestion reaction supernatants were directly pooled for MALDI-TOF mass spectrum. After collecting the supernatant, the digested gel pieces were extracted three times by sonication for 5 min in 60% acetonitrile, 0.1% trifluoroacetic acid, and 0.1% n-octyl-β-d-glucopyranoside. These extracts were combined and dried in a SpeedVac. The dried sample was dissolved in 5 μl of 50% acetonitrile, 0.1% trifluoroacetic acid, sonicated for 2 min, and used for recording the MALDI-TOF mass spectrum. Chymotrypsin digestion was as above, with the exception that digestion was performed for 5 h and the reaction was stopped by adding trifluoroacetic acid to a final concentration of 5%. Reaction supernatant and extracts were combined and concentrated to ∼20 μl by SpeedVac. The pH was adjusted by NH4HCO3 if needed. Reduced samples were prepared by adding freshly prepared TCEP to a final concentration of 2 mm to the non-reduced samples. MALDI-TOF Mass Spectrometry—0.3 μl of the above sample and 0.3 μl of 10 mg/ml 1-cyano-4-hydroxysuccinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid (v/v) were deposited on the target plate and air-dried. MALDI mass spectra were recorded with an ABI 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA) MALDI-TOF/TOF mass spectrometer. A frequency-tripled Nd:YAG laser ionized samples at a pulse frequency of 200 Hz with its power adjusted between 15 and 30 μJ, depending on the sample. Laboratory air was used as the collision gas and the collision cell vacuum pressure was 7–9 × 10–7 Torr. MALDI-TOF data from tryptic digests were calibrated with auto-proteolytic peaks (internal standards) and mass errors were less than 20 ppm. The mass range for TOF MS scan functions was set to m/z 500 to 4000. TOF MS/MS scan functions were calibrated externally against the fragments of either angiotensin I or adrenocorticotropic hormone fragment 18–39 depending upon the precursor mass. The mass accuracy of CID data was typically better than 50 ppm. Limited Trypsinization of Carboxylase—Limited trypsin digestions of carboxylase in the presence of propeptide were performed as previously described (23Wu S.M. Mutucumarana V.P. Geromanos S. Stafford D.W. J. Biol. Chem. 1997; 272: 11718-11722Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A pilot reaction was run for each batch of trypsin and carboxylase to determine the optimal conditions for limited cleavage. Cleavage was accomplished on ice in the presence of 5 μm proFIX. Trypsin cleavage was stopped by the addition of H-d-Phe-Pro-Arg chloromethylketone and Aprotinin to a final concentration of 1.6 μm. Samples were subjected to a reducing and non-reducing gradient (4–20%) SDS-PAGE, and the protein bands were made visible through silver staining. Peptide Mass Fingerprint of Human Vitamin K-dependent Carboxylase by MALDI-TOF MS—Freshly purified carboxylase (37Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar) was deglycosylated, fractionated by SDS-PAGE, and digested in-gel (30van Montfort B.A. Canas B. Duurkens R. Godovac-Zimmermann J. Robillard G.T. J. Mass Spectr. 2002; 37: 322-330Crossref PubMed Scopus (80) Google Scholar). Fig. 1 shows a typical MALDI-TOF mass spectrum of the trypsin-digested non-reduced carboxylase. The MS-Fit search program (38Clauser K.R. Baker P. Burlingame A.L. Anal. Chem. 1999; 71: 2871-2882Crossref PubMed Scopus (981) Google Scholar), a part of "Protein Prospector" Version 4.0.4 (available at prospector.ucsf.edu), was used to identify the mass peaks in the "NCBInr.5.28.2003" data base. The results of the search are shown in Table I. Cysteine-containing peptides are marked by an asterisk in Fig. 1. A diagram of the sequence coverage of carboxylase by MALDI-TOF MS is shown in Fig. 2. For trypsin digestion, 38.8% of the expected peptides were recovered; this coverage could be increased to 41.9% by re-extraction of the gel pieces after the supernatant had been removed.Table IAssignment of fragment ions obtained from MALDI-TOF MS of the in-gel trypsin digestion of human vitamin K-dependent γ-glutamyl carboxylaseResiduesMeasured massExpected massm/zm/z407-412735.3536735.390284-90747.3871747.4001666-671787.4305787.4314674-680900.4328900.4328—1-9906.4529906.4467446-453aCysteine-containing peptides.941.4496941.4515327-334952.6205952.6195319-326aCysteine-containing peptides.965.4939965.4879319-326aCysteine-containing peptides.,bAcrylamide modified cysteine residue containing peptide.1036.52761036.525092-100aCysteine-containing peptides.1053.50461053.5039673-6801056.54611056.5339235-2421061.55371061.557320-291085.60001085.595592-100aCysteine-containing peptides.,bAcrylamide modified cysteine residue containing peptide.1124.55021124.5410437-445cPeptides recovered from the gel extract.1143.54221143.5621579-5881173.58361173.5727335-346aCysteine-containing peptides.1249.61671249.6251648-6611511.82301511.8222486-4981516.79981516.7912192-2041552.84671552.838937-491609.80481609.8015420-4351738.87841738.876469-83cPeptides recovered from the gel extract.1824.97421824.9722499-5131828.95611828.9420219-2341933.87371933.864251-681983.14031983.1391218-2342061.95922061.9592689-704aCysteine-containing peptides.,bAcrylamide modified cysteine residue containing peptide.2076.08302076.1251458-476dGlycosylated peptides.2373.19782372.1563625-647dGlycosylated peptides.2433.29032432.2561623-647dGlycosylated peptides.2690.42702689.3936a Cysteine-containing peptides.b Acrylamide modified cysteine residue containing peptide.c Peptides recovered from the gel extract.d Glycosylated peptides. Open table in a new tab Fig. 2Amino acid sequence of human γ-glutamyl carboxylase showing tryptic and chymotryptic peptide fragments detected by MALDI-TOF MS. Boldface residues, recovered from the trypsin digestion supernatant. Residues in boldface italic were recovered from an additional extract of trypsin-digested gel plugs. Underlined residues were recovered from chymotrypsin digestion. Boxed regions represent transmembrane segments according to our recent carboxylase topology study (11Tie J. Wu S.M. Jin D. Nicchitta C.V. Stafford D.W. Blood. 2000; 96: 973-978Crossref PubMed Google Scholar). The sequence coverage for tryptic digestion is 41.9%. Chymotryptic sequence coverage is 30.5%. The combined sequence coverage is 62.3%.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There are three peptide peaks (Table I) at m/z 2373.2, 2433.3, and 2690.4 corresponding to residues 458–476, 625–647, and 623–647 that differ by 1 unit from the expected trypsin-digested fragments. Each of these peptides has a consensus sequence for N-glycosylation. Because deglycosylation by PNGase F converts an asparagine residue to an aspartic acid residue, the 1-unit mass shift is expected. This prediction was confirmed by MS/MS sequencing of the tryptic peptides and deglycosylation of carboxylase in the presence of 18O-labeled water (data not shown). To increase the sequence coverage, limited chymotrypsin digestion of carboxylase was performed. Chymotrypsin digestion alone covers 30.5% of the carboxylase sequence, some of which overlaps with the sequence recovered by trypsin digestion (Fig. 2). Total sequence coverage with trypsin and chymotrypsin was 62.3% of the entire carboxylase. Higher sequence coverage is difficult to obtain because the hydrophobic peptide region and the membrane-spanning segments are either not readily accessible to proteolytic enzymes, and/or are difficult to extract from the gel pieces. As shown in Fig. 2, peptides containing all of the ten cysteines were recovered. Disulfide-linked Peptides of the Carboxylase Molecule—The program MS-Fit in Protein Prospector was used to search for possible disulfide-linked peptides in both the trypsin and chymotrypsin digestions of the non-reduced carboxylase samples. The candidate peaks were then compared with the peptides from a MALDI-TOF mass spectrum of the reduced sample. A peptide ion of m/z at 1991.9 (Fig. 3A) from the trypsin-digested non-reduced sample matches the expected size of a peptide consisting of residues 92–100 (m/z at 1053.5) disulfide-linked to residues 446–453 (m/z at 941.4). As would be expected if this peak represented a disulfide-linked peptide, it was not present in the spectrum of the reduced sample (Fig. 3B). This is the only candidate disulfide-linked peptide that disappears in the reduced sample. Therefore, it appears to be the only disulfide bond in the carboxylase because peptides containing all of the ten cysteines were identified (Fig. 2). Small signals of the reduced forms of peptides 92–100 (m/z at 1053.5) and 446–453 (m/z at 941.4) were also observed in the non-reduced trypsin-digested carboxylase sample (Table I). This is the result of MALDI-induced cleavage of the disulfide bond, as observed by other groups (39Patterson S.D. Katta V. Anal. Chem. 1994; 66: 3727-3732Crossref PubMed Scopus (143) Google Scholar, 40Jones M.D. Patterson S.D. Lu H.S. Anal. Chem. 1998; 70: 136-143Crossref PubMed Scopus (87) Google Scholar, 41Crimmins D.L. Saylor M. Rush J. Thoma R.S. Anal. Biochem. 1995; 226: 355-361Crossref PubMed Scopus (61) Google Scholar, 42Zhou J. Ens W. Poppe-Schriemer N. Standing K.G. Westmore J.B. Int. J. Mass Spectr. Ion Processes. 1993; 126: 115-122Crossref Scopus (42) Google Scholar, 43Happersberger H.P. Bantscheff M. Barbirz S. Glocker M.O. Methods Mol. Biol. 2000; 146: 167-184PubMed Google Scholar). In addition, there is a small amount of free cysteine 99 and 450 in the non-reduced sample vide infra. MS/MS Analysis of Peptide m/z at 1991.9 —We isolated the peptide consisting of residues 92–100 disulfide-linked to residues 446–453 at m/z 1991.9 by a timed ion selector as the precursor ion for further MS analysis (Fig. 4).