Mass Spectrometric Characterization of Full-length Rat Selenoprotein P and Three Isoforms Shortened at the C Terminus

Selenoprotein P is an abundant extracellular glycoprotein. Its mRNA contains 10 UGAs in an open reading frame terminated by a UAA. This predicts that full-length selenoprotein P will contain 10 selenocysteine residues. Full-length selenoprotein P and three smaller isoforms that have identical N termini have been demonstrated. Selenoprotein P was purified from rat plasma, and the four isoforms were separated by heparin chromatography and SDS-PAGE. Mass spectrometric peptide analysis of the full-length isoform verified 357 of its 366 predicted amino acid residues, including its C terminus and all 10 selenocysteines. The C termini of the smaller isoforms were characterized by mass spectrometry. The shortened isoforms terminated where the second, third, and seventh selenocysteine residues were predicted to be. This suggests that all isoforms arise from the same mRNA and that the UGAs that specify the second, third, and seventh selenocysteines in full-length selenoprotein P can alternatively serve to terminate translation, producing the shorter isoforms. Selenoprotein P is an abundant extracellular glycoprotein. Its mRNA contains 10 UGAs in an open reading frame terminated by a UAA. This predicts that full-length selenoprotein P will contain 10 selenocysteine residues. Full-length selenoprotein P and three smaller isoforms that have identical N termini have been demonstrated. Selenoprotein P was purified from rat plasma, and the four isoforms were separated by heparin chromatography and SDS-PAGE. Mass spectrometric peptide analysis of the full-length isoform verified 357 of its 366 predicted amino acid residues, including its C terminus and all 10 selenocysteines. The C termini of the smaller isoforms were characterized by mass spectrometry. The shortened isoforms terminated where the second, third, and seventh selenocysteine residues were predicted to be. This suggests that all isoforms arise from the same mRNA and that the UGAs that specify the second, third, and seventh selenocysteines in full-length selenoprotein P can alternatively serve to terminate translation, producing the shorter isoforms. More than 15 animal selenoproteins have been characterized, and all of them contain selenocysteine. Selenocysteine incorporation is specified by a UGA in the open reading frame of the mRNA that is accompanied by a “selenocysteine insertion sequence” element in the 3′-untranslated region (1.Berry M.J. Banu L. Chen Y. Mandel S.J. Kieffer J.D. Harney J.W. Larsen P.R. Nature. 1991; 353: 273-276Crossref PubMed Scopus (515) Google Scholar). This is an alternative function of the UGA codon, which usually terminates translation. Selenoprotein P is an unusual extracellular glycoprotein that has been suggested to serve in oxidant defense (2.Burk R.F. Hill K.E. Awad J.A. Morrow J.D. Kato T. Cockell K.A. Lyons P.R. Hepatology. 1995; 21: 561-569PubMed Google Scholar) and in selenium transport (3.Motsenbocker M.A. Tappel A.L. Biochim. Biophys. Acta. 1982; 719: 147-153Crossref PubMed Scopus (160) Google Scholar,4.Burk R.F. Hill K.E. Read R. Bellew T. Am. J. Physiol. 1991; 261: E26-E30Crossref PubMed Google Scholar). It was originally purified from rat plasma, where it is relatively abundant (30 μg of peptide/ml plasma) (5.Read R. Bellew T. Yang J.-G. Hill K.E. Palmer I.S. Burk R.F. J. Biol. Chem. 1990; 265: 17899-17905Abstract Full Text PDF PubMed Google Scholar). The purified protein contained 7.5 ± 1 atoms of selenium per molecule in the form of selenocysteine (5.Read R. Bellew T. Yang J.-G. Hill K.E. Palmer I.S. Burk R.F. J. Biol. Chem. 1990; 265: 17899-17905Abstract Full Text PDF PubMed Google Scholar). The mRNA of selenoprotein P (deduced from its cDNA) contains 10 UGAs in its open reading frame, implying 10 selenocysteines in the primary structure of the protein (6.Hill K.E. Lloyd R.S. Yang J.-G. Read R. Burk R.F. J. Biol. Chem. 1991; 266: 10050-10053Abstract Full Text PDF PubMed Google Scholar). Thus, not all the predicted selenium was detected by analysis of the purified protein. This variance might be due to the presence of isoforms of the protein that contain different numbers of selenocysteine residues or to the absence of selenium from some of the predicted selenocysteine sites. Isoforms of selenoprotein P are present in rat plasma, having been demonstrated by their differential binding to heparin-Sepharose (7.Chittum H.S. Himeno S. Hill K.E. Burk R.F. Arch. Biochem. Biophys. 1996; 325: 124-128Crossref PubMed Scopus (60) Google Scholar). Two isoforms have been purified and characterized by amino acid analysis, conventional peptide sequencing, and C-terminal sequencing (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Both of these isoforms have the same N-terminal amino acid sequence. One of them was shown to be the full-length protein, terminating at a “hard stop” UAA in the mRNA, and the other was shown to terminate at the predicted position of the second selenocysteine, i.e. the second UGA in the open reading frame of the mRNA (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Two other isoforms were identified in that study, but they could not be purified in quantities sufficient for conventional amino acid sequencing. Aside from the demonstration that both of them had the same N-terminal amino acid sequence as the two isoforms that had been purified, these two isoforms have not been characterized. The present study uses mass spectrometry to identify the C-terminal peptides of the two previously uncharacterized isoforms, revealing that they terminate at positions in the sequence where selenocysteine residues are predicted (UGAs). Mass spectrometry was also used to verify the amino acid sequence of the full-length isoform and to confirm that all 10 selenocysteines are present. Iodoacetamide and dithiothreitol were obtained from Sigma. Sequence-grade trifluoroacetic acid was purchased from Burdick and Jackson (Muskegon, MI). The modified N-tosyl-l-phenylalanylchloromethyl ketone-treated porcine trypsin was obtained from Promega (Madison, WI). The other endoproteinases, Lys-C, Glu-C, and Asp-N, were purchased from Roche Molecular Biochemicals. The deglycosylation kit, containing N-glycosidase F, Endo-O-glycosidase, and sialidase A, was obtained from ProZyme (San Leandro, CA). Mass calibration standards, des-Arg1-bradykinin, neurotensin, bovine insulin, melittin, trypsinogen, and bovine serum albumin were purchased from Sigma. Matrix materials, CHCA 1The abbreviations and trivial names used are: CHCA, α-cyano-4-hydroxy-cinnamic acid; sinapinic acid, 3,5-dimethoxy-4-hydroxy cinnamic acid; MALDI, matrix-assisted laser desorption ionization; HPLC, high-performance liquid chromatography; TOF, time of flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; HPLC, high pressure liquid chromatography; MW, molecular weight. and sinapinic acid, were purchased from Aldrich. 75Se-labeled selenite (800 mCi/mg) was purchased from the University of Missouri Research Reactor Facility (Columbia, MO). Other reagents used were analytical grade or better. Rat plasma from which selenoprotein P was purified was purchased from Harlan Bioproducts for Science (Indianapolis, IN). Purification was accomplished as described previously, using a column prepared with the monoclonal antibody 8F11 (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). A heparin-Sepharose column was used to separate selenoprotein P isoform peaks from the purified protein preparation as was done previously (7.Chittum H.S. Himeno S. Hill K.E. Burk R.F. Arch. Biochem. Biophys. 1996; 325: 124-128Crossref PubMed Scopus (60) Google Scholar). These peaks, 1a, 1b, and 2, were used for further characterization. Each separated peak was deglycosylated using N-glycosidase F, endo-O-glycosidase, and sialidase A in a 48-h protocol suggested by the kit manufacturer. SDS-PAGE was performed on the deglycosylated selenoprotein P isoform peaks as described previously (7.Chittum H.S. Himeno S. Hill K.E. Burk R.F. Arch. Biochem. Biophys. 1996; 325: 124-128Crossref PubMed Scopus (60) Google Scholar). The protein gel band of interest was excised from the gel and digested with modified trypsin (9.Rosenfeld J. Capdevielle J. Guillemot J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1121) Google Scholar, 10.Hellman U. Wernstedt C. Gonez J. Heldin C.H. Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (684) Google Scholar). The destained gel slice was washed, crushed, reduced in dithiothreitol, and alkylated with iodoacetamide. The gel pieces were washed, dehydrated in acetonitrile, and swollen in 10 μl of 50 mm ammonium bicarbonate containing trypsin in a concentration of 0.1 μg/μl. After 15 min, additional 50 mm ammonium bicarbonate was added to cover the gel pieces, and digestion was allowed to proceed overnight. After digestion, ∼1 μl of the supernatant was removed for MALDI mass spectrometry analysis. The remaining supernatant was removed, and the gel slices were extracted twice with 50 μl of 60% acetonitrile/0.1% trifluoroacetic acid. These extraction mixtures were dried and combined with the supernatant for injection onto a C18 column. In-solution Glu-C digestion of deglycosylated full-length selenoprotein P isoform (peak 2) was performed in 25 mm ammonium bicarbonate. Approximately 10 μg of protein was reduced in 10 μl of 45 mmdithiothreitol and alkylated with iodoacetamide. Then 0.3 μg of Glu-C was added before incubation at 37 °C for 18 h. The digestion mixtures were neutralized with 1 μl of 5% trifluoroacetic acid, and peptides were separated on a reverse-phase C18 column. The C-terminal peptide of each isoform of selenoprotein P was isolated from a proteolytic digestion with different endoproteinases. Approximately 10 μg of each selenoprotein P isoform peak was reduced and alkylated before enzymatic digestion as described above. Peak 1a was digested with 0.2 μg of Asp-N in phosphate buffer (pH 8.0) for 18 h, and another sample of it was digested with 0.2 μg of modified trypsin in 50 mm ammonium bicarbonate for 18 h. Peak 1b was digested with 0.2 μg of Lys-C in 50 mmTris-HCl buffer (pH 8.5), and peak 2 was digested with 0.25 μg of Glu-C in 25 mm ammonium bicarbonate. The digestion mixtures were neutralized with 1 μl of 5% trifluoroacetic acid and separated on a C18 column to isolate the C-terminal peptides. The proteolytic peptide mixtures were separated on a Vydac microbore C18 column (2.1 × 250 mm) (Vydac, Hesperia, CA) using an HP 1100 HPLC system (Hewlett-Packard Co., Wilmington, DE). The separation was monitored with a UV detector at 214 nm. Solvent systems of A (0.1% trifluoroacetic acid) and B (94.9% acetonitrile/5% H2O/0.1% trifluoroacetic acid) were used. The peptides were eluted from the column with a flow rate of 0.2 ml/min using the following gradient program: 5% solvent B, 10 min, isocratic; 5–60% solvent B over 50 min, linear gradient; 60% solvent B, 10 min, isocratic; 60–80% solvent B in 10 min, linear gradient; 80% solvent B, 10 min, isocratic; then to 5% solvent B in 5 min, linear gradient. UV-monitored peptide peaks were manually collected in Eppendorf safe-lock polypropylene microcentrifuge tubes. HPLC fractions were lyophilized dry and reconstituted in 40 μl of 50% acetonitrile/50% H2O/0.1% trifluoroacetic acid for mass spectrometry analysis. MALDI mass spectra were obtained using a Perseptive DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a 337-nm nitrogen laser. The instrument was operated in the linear mode under optimized delayed extraction conditions for peptide and protein analysis. The selenium-containing peptides were analyzed in the reflector mode to obtain high resolution to identify the selenium isotope distribution. Mass calibration was accomplished using des-Arg1-bradykinin (MW 903.46) and bovine insulin (MW 5733.58) for peptide analysis and bovine trypsinogen (MW 23981) and bovine serum albumin (MW 66430) for protein analysis in the linear mode. In reflector mode, neurotensin (MW 1671.909) and melittin (MW 2844.754) were used as calibration compounds. A matrix of CHCA, prepared at 10 mg/ml in 50% acetonitrile/49.9% H2O/0.1% trifluoroacetic acid, was used for peptide analysis, and sinapinic acid at 10 mg/ml in 50% acetonitrile/49.9% H2O/0.1% trifluoroacetic acid was used as the matrix for protein analysis. The samples were prepared by dried-droplet method on a stainless steel MALDI plate. Nanoelectrospray was performed on a Finnigan LCQ (Finnigan, San Jose, CA) with a nanospray ion source installed (Protana A/S, Odense, Denmark). The sample of 2 μl was loaded into the metal-coated glass capillary (Protana A/S, Odense, Denmark). The capillary was positioned about 1 mm from the heated capillary. The spray voltage was set at 800 V. The heated capillary was kept at 150 °C. The capillary voltage was set at 43 V, and the tube lens was offset at −10 V. The other parameters for ion optics were tuned to obtain the most intensity for the ion of interest. In MS/MS mode, the precursor ion of interest was isolated and fragmented at 25–35% collision energy, depending on the nature of the peptide. Theoretical isotope peaks were calculated using software that was supplied with the Perseptive DE-STR MADLI-TOF mass spectrometer. Mass spectrometric peptide mapping has been an important analytical tool for verifying protein sequence and identifying proteins by data base search. This technique involves the digestion of the protein by specific endoproteases or chemicals. The resulting peptide mixture is subjected to analysis by mass spectrometry, for example MALDI-TOF MS. The comparison of the set of peptide molecular ion masses observed with the expected digestion fragment masses generally yields a sequence coverage of 60–90%. Using this technique, we have verified the sequence of selenoprotein P, originally deduced from its cDNA. Fig. 1 shows peptide maps of the full-length selenoprotein P isoform produced by MALDI-TOF MS after trypsin and Glu-C digestion. The masses of peaks in the spectra were compared with theoretical masses of predicted peptides (Tables S1 and S2 in the on-line supplement), and the peaks in the spectra that could be identified were labeled with the residue numbers of the peptides. Only three stretches of three amino acid residues each were not verified in this study. The sequence coverage was 97.5%, and it is shown in Fig. S1 in the on-line supplement. Selenocysteine was confirmed to be present in the peptides predicted to contain it by showing that the determined peptide mass matched the predicted peptide mass the mass of the peptide the 10 selenocysteines at residues and have been the that identified each of the selenocysteine residues in the full-length of the of the 10 selenocysteine residues using MALDI-TOF mass spectrometry operated in the reflector of selenocysteine of the and selenocysteine residues were modified by is for the isotope peak of the peptide that has the is the and selenocysteine residues were modified by This is for the isotope peak of the peptide that has the in a Mass is In selenium was identified in peptides by its isotope has a isotope Fig. 2 shows the of the mass peak of a peptide to residues of selenoprotein P. Fig. shows the peak of this from MALDI-TOF mass spectrometry operated in reflector mode. Fig. shows the theoretical peak of this that was by a Fig. shows the theoretical peak of the same peptide with a the selenium In Fig. and are Fig. has a different mass This the that the peptide contains selenium This was further by the peptide ion at to verify the peptide sequence The shows the of the which the of and its of selenium the at the C-terminal as a the abundant ion was It is of interest to that the ion in this was a ion due to the at the N The of the predicted sequence of the peptide. peaks of containing and four selenium atoms have isotope S2 in the on-line The isotope of each peak the predicted by a peaks containing and four selenium shown in Fig. in the on-line the in further using MS/MS to verify the predicted amino acid sequence as as in the on-line The of confirmed the predicted sequence of each of the selenoprotein P can be separated into three peaks using heparin chromatography (7.Chittum H.S. Himeno S. Hill K.E. Burk R.F. Arch. Biochem. Biophys. 1996; 325: 124-128Crossref PubMed Scopus (60) Google Scholar). Two of the peaks, 1b and 2, contain isoform of the selenoprotein The other 1a, contains two isoforms, and these have not been characterized the demonstration that they have the same N terminus as the other two isoforms. MALDI-TOF mass spectra of the three peaks from the heparin column were obtained in the on-line Peak 2 is the full-length isoform and has an mass of ± Peak 1b is the isoform and has an mass of ± Peak 1a two peaks with masses of ± and ± which are both in the other two isoforms. This that peak 1a contains two additional isoforms. four mass peaks are which is with that is the of their the for the C termini of the isoforms. Each isoform was digested with a and the predicted C-terminal peptide was using mass spectrometry. After of the peak by MALDI-TOF mass spectrometry, the amino acid sequence was determined by MS/MS to confirm that it was the predicted C-terminal peptide. The C-terminal peptide of the isoform was identified in a Lys-C digestion, and its amino acid sequence was verified by MS/MS in the on-line This the of this isoform that was done by conventional C-terminal sequencing (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). A in the on-line confirmed that the isoform in peak 2 was on the mass of the protein by MALDI-TOF MS, the shorter isoform in peak 1a to terminate at the UGA in the confirm peak 1a was subjected to Asp-N digestion. The C-terminal peptide was isolated by HPLC and was subjected to MS/MS by the ion with to verify the as shown in Fig. These confirm that this isoform terminates at the UGA The isoform in peak 1a was predicted to terminate at the seventh or UGA on its mass of the on-line a trypsin of it was by MALDI-TOF MS predicted C-terminal peptide could be the was subjected to HPLC, and each peak was by MALDI-TOF MS. Fig. 5 shows the of HPLC In Fig. a is with the mass by at mass of and another These mass to the masses of and acid, This that this peptide is an The was further with and sialidase The and the intensity of the peptide peak at This is the predicted mass of the C-terminal peptide that terminates at the seventh MS/MS of the sequence of the deglycosylated peptide was obtained of the on-line The of that shows the of the ion which that the peptide contains selenocysteine residue These that this peptide is and that the isoform terminates at the seventh sequencing of the shorter three isoforms was not in this study, but some of their peptides were identified by mass not in the performed to their C selenoprotein of the predicted amino acid sequence was identified by selenoprotein and selenoprotein and of the predicted amino acid residues were Thus, four isoforms of selenoprotein P have been the same N terminus and to be from the same amino acid sequence. One isoform is but the other three terminate at positions predicted to be by selenocysteine residues in the open reading frame of the Selenoprotein P is the selenoprotein identified that contains than selenium The presence of 10 UGAs in the open reading frame of rat selenoprotein P mRNA that the full-length protein contains 10 selenocysteine residues. is for selenocysteines in a protein and it is at that a UGA might specify incorporation of a residue other than selenocysteine, it important to selenocysteine was present at the sequence position of each In we had shown that the selenocysteines are present at their predicted positions (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). that all 10 selenocysteines are present in the full-length protein. of the selenocysteines had been This that all are for Selenoprotein P, as purified from rat plasma, is present as four isoforms. One isoform is selenoprotein it terminates at the UAA in the mRNA and contains 10 selenium atoms The other three isoforms terminate at positions of UGA in the open reading frame, where selenocysteine is present in selenoprotein the shorter isoforms terminate at the second, third, and seventh suggests that all four isoforms the same amino acid sequence and from another by terminating at different that sequence A study this for selenoprotein and selenoprotein (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Each of two isoforms was shown to have the expected amino acid and and C-terminal amino acid amino acid sequencing of a mixture of all isoforms by predicted by the (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the present study, the high of mass spectrometry allowed further of all four isoforms. The amino acid sequence of selenoprotein was demonstrated to the predicted sequence with amino acid residues not verified S1 of the on-line residues, had been verified in the study (8.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google four and of the of 366 predicted by analysis of the protein. of the of the three shorter isoforms were verified These are with the that all four isoforms arise from the same The nature of UGA as a and a has been before by M.J. U. S. 1995; PubMed Scopus Google Scholar, S. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Harney J.W. M.J. J. Biol. PubMed Scopus Google Scholar). Using with selenoprotein these have shown that the of the UGA its The two and the from the UGA have In the systems a in the position M.J. U. S. 1995; PubMed Scopus Google Scholar). The second, and UGAs in selenoprotein P mRNA are by A has shown that the can also a is by a R. Harney J.W. M.J. J. Biol. PubMed Scopus Google Scholar). In selenoprotein P, the third, and UGA are by a Thus, all 10 UGA of selenoprotein P mRNA are present in in the have generally S. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and that is not to the present Thus, of the of the isoforms in this study. It that that can be produced of the mRNA will be for the to isoform Selenoprotein P a to study in in the alternative of the UGA It a theoretical that the shorter isoforms are produced from the full-length protein by that were the have to be a that the protein at specific selenocysteine residues. is it that of is for of the isoforms. This The for and of selenoprotein P not the isoforms. This suggests that the are in the N-terminal region of the protein. It will be important to to and isoforms of selenoprotein P. will of the that each isoform and of the of each of R.F. Hill K.E. Read R. Bellew T. Am. J. Physiol. 1991; 261: E26-E30Crossref PubMed Google Scholar, R.F. Hill K.E. Chittum H.S. Biol. PubMed Scopus Google Scholar, T. Read R. J. Burk R.F. Am. J. Physiol. 1992; PubMed Google Scholar) with the molecular of the isoforms of selenoprotein P into their J. for S. and for preparation of selenoprotein P and of the protein for sequencing. with