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Protein Science (2003), 12:2320-2326.
Copyright © 2003 The Protein Society

Detection of four oxidation sites in viral prolyl-4-hydroxylase by top-down mass spectrometry

Ying Ge, Brian G. Lawhorn, Mariam ElNaggar, Siu Kwan Sze, Tadhg P. Begley and Fred W. McLafferty

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA

Reprint requests to: Fred W. McLafferty, Department of Chemistry and Chemical Biology, Cornell University, Baker Laboratory, Ithaca, NY 14853-1301, USA; e-mail: fredwmcl{at}aol.com; fax: (607) 255-7880.

(RECEIVED May 9, 2003; FINAL REVISION July 10, 2003; ACCEPTED July 11, 2003)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03244403.


   Abstract
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 Abstract
Introduction
 Results
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 References
 
Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 ± 3, and <2%, respectively; "top-down" MS/MS of these ions show oxidation at the sites R28–V31, E95–F107, and K216 of 22%, 28%, and 34%, respectively, but with a possible (~4%) fourth site at V125–D150. However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable ~13% oxygen at the V125–D150 site. For the "bottom-up" approach, detection of the ~4% oxidation at the V125–D150 site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just ~4% of this, but the uniqueness of its mass value (~1–2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V125–D150 until K216 is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K216, H137, and D139 are conserved residues.

Keywords: Protein oxidation; top-down mass spectrometry; prolyl 4-hydroxylase; 5-oxaproline


   Introduction
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Mass spectrometry (MS) now provides accurate and efficient identification of an unknown protein from among the thousands predicted by the DNA sequence of the genome (Mann and Wilm 1994; Andersen et al. 1996; Schey and Finley 2000; Smith 2000; Yates III 2000; Aebersold and Goodlett 2001). For routine "bottom-up" identification, proteolysis of a purified protein yields a complex peptide mixture, from which accurate molecular weights of a relatively few peptides by MS (or of peptide fragments by MS/MS) are often sufficient for identification of the protein with high confidence. However, this identification is independent of, and mostly oblivious to, any DNA sequence errors or posttranslational modifications of the protein; peptides whose mass values do not fit the predicted sequence are common from impurities. In addition, complete coverage of the protein sequence by identified peptides is rare (Andersen et al. 1996; Schey and Finley 2000; Smith 2000; Yates III 2000). In the contrasting "top-down" approach (Kelleher et al. 1999a), MS of even impure samples gives mass-isolated "highly pure" molecular ions of the target protein, whose accurate molecular weight value indicates the presence of DNA sequence errors and/or posttranslational modifications. MS/MS dissociation of these ions then yields fragment masses that can only be from the protein: Those not predicted for the unmodified sequence can then identify the locations of even multiple modifications (Kelleher et al. 1998, 1999b; Wilkins et al. 1999; Fridriksson et al. 2000; Kelleher 2000; Meng et al. 2001; Ge et al. 2002; Sze et al. 2002, Sze et al. 2003). Here, the top-down approach is applied to an oxidatively inactivated prolyl-4-hydroxylase (P4H) to detect and localize four sites of oxidation, despite the fact that the sample contained less than one atom of oxygen per molecule, on average. This surely would have been impractical with the bottom-up approach.

P4H catalyzes the hydroxylation of proline residues in pro-collagens and other proteins that contain collagen-like sequences (Scheme 1Go; Prockop and Kivirikko 1995). This reaction requires Fe2+, molecular O2, {alpha}-ketoglutarate, and ascorbate as cosubstrates (Prockop and Kivirikko 1995; Kivirikko and Pihlajaniemi 1998), and plays a critical role in the biosynthesis of collagen because 4-hydroxyprolyl residues stabilize the collagen triple-helix. The viral P4H, whose self-oxidation is characterized here, was recently cloned from Paramecium Bursaria Chlorella Virus (PBCV). This enzyme shows sequence similarity to the C-terminal half of the catalytic {alpha}-subunit of mammalian P4H (Eriksson et al. 1999).



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  		Scheme 1
 
An oxidized protein is often heat-labile and of low catalytic activity (Berlett and Stadtman 1997; Dean et al. 1997; Shacter 2000), and its accumulation is associated with aging and disease (Stadtman 1992; Berlett and Stadtman 1997; Dean et al. 1997). Many nonheme iron-dependent enzymes undergo oxidative self-inactivation (Que Jr. and Ho 1996; Solomon et al. 2000; Dove and Klinman 2001), but the molecular details of this inactivation are unclear. Barlow et al. have proposed a mechanism for the inactivation of 1-aminocyclopropane-1-carboxylate oxidase (Barlow et al. 1997), while Liu et al. have demonstrated that the inactivation of TfdA occurs by hydroxylation of Trp112 based on the analysis by nanoscale LC/MS/MS of tryptic peptides (bottom-up approach; Liu et al. 2001). In contrast to their analytical strategy, we report here the analysis of oxidized viral P4H starting directly with the intact protein (top-down approach).


   Results
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MS analysis of oxidized P4H
The oxidation of viral P4H that occurs during a 12-h incubation in the presence of peptide substrates was analyzed by ESI/MS. The P4H molecular ions (Fig. 1aGo) give a distribution of isotopic peaks 1 Dalton apart, whose abundances represent the probability of containing heavier isotopes such as 13C, 15N, etc. The mass of the most abundant isotopic peak, 25,686.7-16, agrees with the molecular weight (Mr) value, 25,686.7-16, calculated for the recombinant P4H (considering the loss of the N-terminal methionine and the presence of two disulfide bonds). In the spectra of the incubated samples, the next clusters, Mr = 25,702.9-16 and 25,718.8-16, are 16 and 32 Daltons heavier, corresponding to the addition of one and two oxygen atoms. One spectrum (Fig. 1fGo) clearly indicates that at least three oxygen atoms (+48 Daltons) can be added to P4H.



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  		Figure 1. Partial ESI/MS spectrum of viral P4H (25+ ions) after 12-h incubation with substrates: a,b, Pro-Ala-Pro-Lys; c, (Pro-Ala-Pro-Lys)2; d, (Pro-Ala-Pro-Lys)3; e, Pro-Ala-OxaPro-Lys; f, Pro-Ala-Pro-Lys-OxaPro-Ala-Pro-Lys-Pro-Ala-Pro-Lys. All but a are with ascorbate.

 
Removal of the essential cosubstrate, ascorbate, from the reaction mixture essentially stops the oxidation of P4H (Fig. 1aGo), indicating a direct relationship between protein oxidation and P4H enzymatic activity. For the (Pro-Ala-Pro-Lys)n substrates, that for n = 1 (Fig. 1bGo) shows abundances of 28% O1 adducts (+16 Daltons), 12% O2, and ~2% O3, and the average number of oxygen atoms per P4H molecule is 0.58 (0.28 + 2 x 0.12 + 3 x 0.02). Increasing the substrate size to n = 2 and n = 3 increases this value to 0.95 and 0.94, respectively. This also suggests that protein hydroxylation is associated with catalysis because the longer peptides are better substrates for the enzyme (Eriksson et al. 1999).

We have previously demonstrated that oxaproline-containing peptides inactivate human P4H by forming a stable peptide enzyme adduct (Günzler et al. 1988; Wu et al. 1999). We were unable to identify the labeled residue for this interesting inactivation reaction due to intractable solubility problems with the {alpha}-subunit of the human enzyme. The high quality spectra that we were able to obtain here prompted us to repeat this experiment with the viral enzyme. We synthesized Pro-Ala-OxaPro-Lys and Pro-Ala-Pro-Lys-OxaPro-Ala-Pro-Lys-Pro-Ala-Pro-Lys, and tested both peptides as mechanism-based inactivating agents. Although both peptides stimulated the hydroxylation of the viral enzyme (Fig. 1e,fGo), MS analysis demonstrated that neither peptide covalently labeled the protein.

The time dependence of the oxidation of P4H in the presence of (Pro-Ala-Pro-Lys)2 provides further evidence (Fig. 2Go) for the nature of the oxidations. Assuming that the protein hydroxylation requires catalytically competent enzyme, the time course data, as well as the data in Figure 1Go, suggest that monohydroxylated and dihydroxylated enzyme are both able to catalyze a further protein hydroxylation. In addition, the observation that all three hydroxylation reactions are occurring at comparable rates suggests that a combination of active site-directed oxidation and oxidation mediated by a reactive oxygen species that has diffused out of the active site is unlikely because such oxidations should show very different rates. The observation that protein oxidation occurs in buffer containing 2 mM ascorbate and 0.1 mM DTT, and that the enzyme is only partially protected by addition of high concentrations of catalase and superoxide dismutase (12P data, Fig. 2Go) suggest that the oxidation events are occurring at the active site. Localization of the hydroxylation sites further clarifies this.



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  		Figure 2. Relative abundance of viral P4H molecular ions versus time of incubation with (Pro-Ala-Pro-Lys)2. 12P: 12 h incubation containing 0.1 mg/mL each of catalase and superoxide dismutase.

 
Identification of the oxidation sites
The mixture of oxidized and nonoxidized molecular ions (Fig. 1Go) was dissociated with IRMPD and ECD (Table 1Go) to locate the oxidation sites. IRMPD (Little et al. 1994) of the molecular ions of Figure 1dGo, showed (Fig. 3Go) oxidation in y12 and many larger y ions but not in y11, y9, or y7. This specifically localizes one oxidation to K216, the twelfth amino acid residue from the C terminus (Fig. 4Go). Oxidation of this site represents 34% of all the +16 Daltons oxidized ions (Table 1Go), averaging the similar values for the C-terminal fragments y12 through y77 (Table 1Go). Three additional IRMPD spectra of the molecular ions from Figure 1Go, b c, and f (data not shown), also identify K216 as one of the oxidation sites.


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  		Table 1. Singly oxidized fragment ions from IRMPD and ECD of Figure 1dGo ions
 


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  		Figure 3. Partial IRMPD spectra of the molecular ions of Figure 1dGo. Circles at peak tops represent the theoretical isotopic distribution.

 


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  		Figure 4. Alignment of the relevant amino acid residues in the recombinant viral P4H (I) with the native viral P4H (II) and human type I P4H (III) (see text). Conserved amino acids, gray vertical bars; proposed Fe2+ and {alpha}-ketoglutarate binding residues of human type I P4H, overhead inverted triangles; in (I), critical IRMPD and ECD fragment ions and the four oxidation sites proposed.

 
IRMPD and ECD (Horn et al. 2000a; Zubarev et al. 2000) spectra of the Figure 1dGo molecular ions also show N-terminal (a•, b, c) fragment ions. Twelve fragments with less than 28 residues (Table 1Go) contain no observable (<0.03 to <0.16) + 6 Dalton oxidized peaks. However, the b31 fragment does contain +16 Dalton oxidized ions, so that oxidation also occurred within residues 28 to 31. The N-terminal fragment ions containing 31 to 94 residues all have a similar 0.22 proportion of (F + 16)n+ ions, so that further oxidation is not appreciable from residues 32 to 94 (Fig. 4Go).

The +16 Dalton peaks of the b124 and y120 (complement of the b107) ions represent 0.50 and 0.38 proportions, respectively, of the (F + 16)n+ species. The sum of these, 0.88, should represent the oxidation of the whole molecule minus any oxidation in their overlap region (residues 108–124), plus some additional for double oxidation that is within the noise level. In good agreement, the Figure 1dGo, abundances predict an average of 0.41 + 2 x 0.19 + 3 x 0.05 = 0.94 oxygen atoms per molecular ion. Thus, there is negligible oxidation from residues 108–124, plus substantial oxidation between residues 95 to 107 (0.50–0.22 = 0.28). For residues 125 to 150, the difference (0.38–0.34 = ~0.04) is close to the value of the experimental error.

Oxidation increase by top-down
To obtain more definitive information on the last oxidation region, the more highly oxygenated (M + 32)22–26+ ions were isolated. From IRMPD of these doubly oxidized ions (Fig. 5Go), again, only non- and mono-oxidized (+16 Daltons) isoforms are observed within 77 residues from the C terminus, and all are consistent with K216 oxidation. However, the doubly oxidized form (+32 Daltons) has increased from ~3% in y77 to 14% in y105 (Fig. 5Go) and y120 (while K216 increased from 34% to 47%), confirming another oxidation location in V125–D150 (Fig. 4Go). In contrast to the first three oxidations (Fig. 2Go), the oxidation rate in the V125–D150 region is greatly increased by oxidation at other sites.



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  		Figure 5. Partial IRMPD spectra of SWIFT isolated [M + 32]22–26+ molecular ions of Figure 1dGo.

 

   Discussion
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Materials and methods
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The top-down MS/MS data described here have identi- fied four oxidation sites in viral P4H: R28–V31, E95–F107, V125–D150, and K216 with relative reactivities (Fig. 1dGo sample) of 5.5:7:1:8.5, respectively. K216 is a conserved residue in the P4H family (Fig. 4Go; Eriksson et al. 1999). It has been proposed, based on mutagenesis, that this lysine binds the C-5 carboxyl group of {alpha}-ketoglutarate (Myllyharju and Kivirikko 1997). This suggests that the oxidation of K216 is occurring at the active site. H137 and D139 are also conserved and the corresponding residues in the human P4H are proposed, based on mutagenesis, to function as ligands to the iron (Myllyharju and Kivirikko 1997). This suggests that the oxidized residue on the V125–D150 peptide may also be located at the active site. In the absence of a structure, it is not possible to localize further the oxidations at E95–F107 or R28–V31, although these also contain conserved residues (Fig. 4Go). Their extents of oxidation (0.22, 0.28) in the P4H sample of Figure 1dGo (0.94 oxygens) are nearly as great as that of K216 (0.34); these relatively high reactivities suggest that these are also likely to be located in the active site.

Although the K216 oxidation identified here required only a single spectrum (Fig. 3Go), this identification would have been complicated with a "bottom-up" approach. The trypsin digest would have produced dozens of peptides in a mass region an order of magnitude smaller than that of the IRMPD/ECD spectra here, complicating the identification and quantitation of the oxidized peptides and, especially, of MS/MS to identify the K216 site. Further, the V125–D150 region had only 0.04 oxygens per molecule of the 0.94 total in the Figure 1dGo precursors, a nearly impossible noise level for identification of a complement +16-Dalton peptide. By selecting only the dioxygenated molecular ions in the top-down approach, this oxygenated site was easily identified (Fig. 5Go, y105 versus y77). Finally, this allowed measurement of kinetic data at two levels of oxidation in the same sample.


   Materials and methods
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 Materials and methods
References
 
Chemicals were purchased from Sigma or Aldrich, and were used without further purification. {alpha}-[1-14C] Ketoglutarate was purchased from ARC. Recombinant viral P4H was overexpressed from plasmid pET30b-PBCV-1E36 and purified on a His-Bind Ni2+-chelate affinity column (Novagen; Eriksson et al. 1999). P4H activity was determined using the hydroxylation-coupled decarboxylation of {alpha}-[1-14C] ketoglutarate (Kivirikko and Myllylae 1982). 5-Oxaproline (OxaPro) containing peptides were synthesized as described previously (Vasella et al. 1983; Wu et al. 1999) and unmodified peptides were prepared using standard peptide synthesis chemistry.

P4H (250 µg) was incubated at 37°C for 1, 3, and 12 h in a 2-mL total reaction mixture containing 50 mM Tris, pH 7.5, 0.1 mM DTT, 2 mM ascorbate, 0.05 mM FeSO4, 0.5 mM {alpha}-ketoglutarate, and 1 mM peptide substrates, Pro-Ala-Pro-Lys, (Pro-Ala-Pro-Lys)2, (Pro-Ala-Pro-Lys)3, Pro-Ala-OxaPro-Lys, and Pro-Ala-Pro-Lys-OxaPro-Ala-Pro-Lys-Pro-Ala-Pro-Lys. The protein was concentrated and desalted into 76:20:4 CH3OH/H2O/CH3COOH using a Centricon 10,000 MWCO centrifugal concentrator (Millipore). This solution was introduced via a nanospray ESI emitter into a modified 6-T Finnigan Fourier-transform (FT) ion cyclotron resonance mass spectrometer (Beu et al. 1993). The outer trapping plates, conventionally used for electron containment (Zubarev et al. 2000), were used for ion trapping. Pulsed nitrogen (~10-6 Torr) was used to assist trapping. For MS/MS spectra, infrared multiphoton dissociation (IRMPD; Little et al. 1994) and "in-beam" activated ion electron capture dissociation (ECD; Horn et al. 2000a) were applied to the protein mixture directly. In some cases, specific ions were isolated using stored waveform inverse FT (Marshall et al. 1985) followed by IRMPD or collisionally activated dissociation (CAD; Gauthier et al. 1991; Senko et al. 1994). The MS/MS spectra were an average of 20–100 scans. Assignment of the fragment masses and compositions were made with the computer program THRASH (Horn et al. 2000b). The mass difference (in units of 1.00235 Daltons) between the most abundant isotopic peak and the monoisotopic peak is denoted in italics after each Mr value.


   Acknowledgments
 
We acknowledge Johanna Myllyharju and Kari Kivirikko for the P4H overexpression plasmid, and HanBin Oh, Neil Kelleher, and David Horn for helpful discussions. NIH grants GM16609 (to F.W.M.) and DK44083 (to T.P.B.) provided generous financial support.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Aebersold, R. and Goodlett, D.R. 2001. Mass spectrometry in proteomics. Chem. Rev. 101: 269–295.[CrossRef][Medline]

Andersen, J.S., Svensson, B., and Roepstorff, P. 1996. Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: Powerful analytical tools in recombinant protein chemistry. Nat. Biotechnol. 14: 449–457.[CrossRef][Medline]

Barlow, J.N., Zhang, Z., John, P., Baldwin, J.E., and Schofield, C.J. 1997. Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications. Biochemistry 36: 3563–3569.[CrossRef][Medline]

Berlett, B.S. and Stadtman, E.R. 1997. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272: 20313–20316.[Free Full Text]

Beu, S.C., Senko, M.W., Quinn, J.P., Wampler III, F.M., and McLafferty, F.W. 1993. Fourier-transform electrospray instrumentation for tandem high-resolution mass spectrometry of large molecules. J. Am. Soc. Mass. Spectrom. 4: 557–565.[CrossRef]

Dean, R.T., Fu, S., Stocker, R., and Davies, M.J. 1997. Biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324: 1–18.

Dove, J.E. and Klinman, J.P. 2001. Trihydroxyphenylalanine quinone (TPQ) from copper amine oxidases and lysyl tyrosylquinone (LTQ) from lysyl oxidase. Adv. Protein Chem. 58: 141–174.[Medline]

Eriksson, M., Myllyharju, J., Tu, H., Hellman, M., and Kivirikko, K.I. 1999. Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates. J. Biol. Chem. 274: 22131–22134.[Abstract/Free Full Text]

Fridriksson, E.K., Beavil, A., Holowka, D., Gould, H.J., Baird, B., and McLafferty, F.W. 2000. Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-d mass spectrometry. Biochemistry 39: 3369–3376.[CrossRef][Medline]

Gauthier, J.W., Trautman, T.R., and Jacobson, D.B. 1991. Sustained off-resonance irradiation for collision-activated dissociation involving Fourier transform mass spectrometry. Collision-activated dissociation technique that emulates infrared multiphoton dissociation. Anal. Chim. Acta 246: 211–225.[CrossRef]

Ge, Y., Lawhorn, B.G., ElNaggar, M., Strauss, E., Park, J.-H., Begley, T.P., and McLafferty, F.W. 2002. Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry. J. Am. Chem. Soc. 124: 672–678.[CrossRef][Medline]

Günzler, V., Brocks, D., Henke, S., Myllyla, R., Geiger, R., and Kivirikko, K.I. 1988. Syncatalytic inactivation of prolyl 4-hydroxylase by synthetic peptides containing the unphysiologic amino acid 5-oxaproline. J. Biol. Chem. 263: 19498–19504.[Abstract/Free Full Text]

Horn, D.M., Ge, Y., and McLafferty, F.W. 2000a. Activated ion electron capture dissociation for mass spectral sequencing of larger (42 kDa) proteins. Anal. Chem. 72: 4778–4784.[Medline]

Horn, D.M., Zubarev, R.A., and McLafferty, F.W. 2000b. Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules. J. Am. Soc. Mass. Spectrom. 11: 320–332.[CrossRef][Medline]

Kelleher, N.L. 2000. From primary structure to function: Biological insights from large-molecule mass spectra. Chem. Biol. 7: R37–R45.[CrossRef][Medline]

Kelleher, N.L., Taylor, S.V., Grannis, D., Kinsland, C., Chiu, H.-J., Begley, T.P., and McLafferty, F.W. 1998. Efficient sequence analysis of the six gene products (7–74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry. Protein Sci. 7: 1796–1801.[Abstract]

Kelleher, N.L., Lin, H.Y., Valaskovic, G.A., Aaserud, D.J., Fridriksson, E.K., and McLafferty, F.W. 1999a. Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry. J. Am. Chem. Soc. 121: 806–812.[CrossRef]

Kelleher, N.L., Zubarev, R.A., Bush, K., Furie, B., Furie, B.C., McLafferty, F.W., and Walsh, C.T. 1999b. Localization of labile posttranslational modifications by electron capture dissociation: The case of g-carboxyglutamic acid. Anal. Chem. 71: 4250–4253.[Medline]

Kivirikko, K.I. and Myllylae, R. 1982. Posttranslational enzymes in the biosynthesis of collagen: Intracellular enzymes. Methods Enzymol. 82: 245–304.

Kivirikko, K.I. and Pihlajaniemi, T. 1998. Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases. Adv. Enzymol. Related Areas Mol. Biol. 72: 325–398.[Medline]

Little, D.P., Speir, J.P., Senko, M.W., O’Connor, P.B., and McLafferty, F.W. 1994. Infrared multiphoton dissociation of large multiply charged ions for biomolecule sequencing. Anal. Chem. 66: 2809–2815.[Medline]

Liu, A., Ho, R.Y.N., Que Jr., L., Ryle, M.J., Phinney, B.S., and Hausinger, R.P. 2001. Alternative reactivity of an a-ketoglutarate-dependent iron(II) oxygenase: Enzyme self-hydroxylation. J. Am. Chem. Soc. 123: 5126–5127.[CrossRef][Medline]

Mann, M. and Wilm, M. 1994. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66: 4390–4399.[Medline]

Marshall, A.G., Wang, T.C.L., and Ricca, T.L. 1985. Tailored excitation for Fourier transform ion cyclotron mass spectrometry. J. Am. Chem. Soc. 107: 7893–7897.[CrossRef]

Meng, F., Cargile, B.J., Miller, L.M., Forbes, A.J., Johnson, J.R., and Kelleher, N.L. 2001. Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat. Biotechnol. 19: 952–957.[CrossRef][Medline]

Myllyharju, J. and Kivirikko, K.I. 1997. Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase. EMBO J. 16: 1173–1180.[CrossRef][Medline]

Prockop, D.J. and Kivirikko, K.I. 1995. Collagens: Molecular biology, diseases, and potentials for therapy. Annu. Rev. Biochem. 64: 403–434.[CrossRef][Medline]

Que Jr., L. and Ho, R.Y.N. 1996. Dioxygen activation by enzymes with mononuclear non-heme iron active sites. Chem. Rev. 96: 2607–2624.[CrossRef][Medline]

Schey, K.L. and Finley, E.L. 2000. Identification of peptide oxidation by tandem mass spectrometry. Acc. Chem. Res. 33: 299–306.[CrossRef][Medline]

Senko, M.W., Speir, J.P., and McLafferty, F.W. 1994. Collisional activation of large multiply charged ions using fourier transform mass spectrometry. Anal. Chem. 66: 2801–2808.[Medline]

Shacter, E. 2000. Quantification and significance of protein oxidation in biological samples. Drug Metab. Rev. 32: 307–326.[CrossRef][Medline]

Smith, R.D. 2000. Evolution of ESI-mass spectrometry and Fourier transform ion cyclotron resonance for proteomics and other biological applications. Int. J. Mass. Spectrom. 200: 509–544.[CrossRef]

Solomon, E.I., Brunold, T.C., Davis, M.I., Kemsley, J.N., Lee, S.K., Lehnert, N., Neese, F., Skulan, A.J., Yang, Y.S., and Zhou, J. 2000. Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem. Rev. 100: 235–349.[CrossRef][Medline]

Stadtman, E.R. 1992. Protein oxidation and aging. Science 257: 1220–1224.[Abstract/Free Full Text]

Sze, S.K., Ge, Y., Oh, H., and McLafferty, F.W. 2002. Top-down mass spectrometry of a 29-kDa protein for characterization of any posttranslational modification to within one residue. Proc. Natl. Acad. Sci. 99: 1774–1779.[Abstract/Free Full Text]

———. 2003. Plasma electron capture dissociation for the characterization of large proteins by top down mass spectrometry. Anal. Chem. 75: 1599–1603.[Medline]

Vasella, A., Voeffray, R., Pless, J., and Huguenin, R. 1983. Synthesis of D- and L-5-oxaproline and a new captopril analog. Helv. Chim. Acta 66: 1241–1252.[CrossRef]

Wilkins, M.R., Gasteiger, E., Gooley, A.A., Herbert, B.R., Molloy, M.P., Binz, P.-A., Ou, K., Sanchez, J.-C., Bairoch, A., Williams, K.L., et al. 1999. High-throughput mass spectrometric discovery of protein post-translational modifications. J. Mol. Biol. 289: 645–657.[CrossRef][Medline]

Wu, M., Moon, H.-S., Begley, T.P., Myllyharju, J., and Kivirikko, K.I. 1999. Mechanism-based inactivation of the human prolyl-4-hydroxylase by 5-oxaproline-containing peptides: Evidence for a prolyl radical intermediate. J. Am. Chem. Soc. 121: 587–588.[CrossRef]

Yates III, J.R. 2000. Mass spectrometry. From genomics to proteomics. Trends Genet. 16: 5–8.[Medline]

Zubarev, R.A., Horn, D.M., Fridriksson, E.K., Kelleher, N.L., Kruger, N.A., Lewis, M.A., Carpenter, B.K., and McLafferty, F.W. 2000. Electron capture dissociation for structural characterization of multiply charged protein cations. Anal. Chem. 72: 563–573.[Medline]


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