Pattern-based algorithm for peptide sequencing from tandem high energy collision-induced dissociation mass spectra.

A new strategy is reported for extracting complete and partial sequence information from collision-induced dissociation (CID) spectra of peptides, CID spectra are obtained from high energy CID of peptide molecular ions on a four-sector tandem mass spectrometer with an electro-optically coupled microchannel array detector, A peak detection routine reduces the spectrum to a list of peak masses and peak heights, which is then used for sequencing, The sequencing algorithm was designed to use spectral data to generate sequence fits directly rather than to use data to test the fit of series of sequence guesses. The peptide sequencing algorithm uses a pattern based on the polymeric nature of peptides to classify spectral peaks into sets that are related in a sequence-independent manner, It then establishes sequence relationships among these sets, Peak detection from raw data takes 10-20 s, with sequence generation requiring an additional 10-60 s on a Sun 3/60 workstation, The program is written in the C language to run on a Unix platform. The principal advantages of our method are in the speed of analysis and the potential for identifying modified or rare amino acids. The algorithm was designed to permit real-time sequencing but awaits hardware modifications to allow real-time access to CID spectra.

Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry.

High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H2N=CH-R](+), where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.

UV light-induced cross-linking of nucleosides, nucleotides and a dinucleotide to the carboxy-terminal heptad repeat peptide of RNA polymerase II as studied by mass spectrometry.

We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross-linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5-bromo-2'-deoxyuridine), two nucleotides (thymidine-5'-monophosphate and 5-bromo-2'-deoxyuridine-5'-monophosphate) and a dinucleotide (thymidylyl-[3'-->5']-2'-deoxyadenosine). The peptide picked was SPSYSPT (L-seryl-L-prolyl-L-seryl-L-tyrosyl-L-seryl-L-prolyl-L-threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed-phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High-energy tandem collision-induced dissociation mass spectrometry pinpointed the location of cross-linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross-linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross-linked DNA-protein complexes.

Probing the binding region of the single-stranded DNA-binding domain of rat DNA polymerase beta using nanosecond-pulse laser-induced cross-linking and mass spectrometry.

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase beta (pol beta-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorption/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol beta-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.

Derivatization of hydrophilic peptides for liquid secondary ion mass spectrometry at the picomole level.

A simple procedure for preparing alkyl and benzyl esters of peptides is described. The procedure can provide an increase in the secondary ion yield of a factor of 25 or more in the liquid secondary ion mass spectra of hydrophilic peptides. The procedure allows rapid in situ derivatization of, e.g., collected, lyophilized HPLC fractions. No sample transfers are required and excess reagents are easily removed. Mass spectrometry of such fractions is typically required to prepare a mass map of the peptides produced by proteolytic digestion of a protein. However, small hydrophilic peptides are often not detected because their low secondary ion yield. Relative yields of MH+ ions from peptides esterified with various alcohols are compared: methanol, 2-propanol, 1-butanol, 1-hexanol, 1-octanol, and benzyl alcohol. The best combination of ion yield and ease of reagent removal is obtained with 1-hexanol. The degree of improvement depends on the specific peptide; the greatest improvement is generally observed with the most hydrophilic peptides. The procedure does not affect side-chain amides. Partial derivatization is sometimes observed with peptides containing more than one carboxyl group. Hexylation is shown to have a leveling effect on the mass spectra of peptide mixtures, allowing detection of surface-inactive peptides in the presence of surface-active ones. Benzyl alcohol is useful for derivatizing peptides that are not retained or that elute very early from reverse-phase HPLC columns. The derivatives have longer retention times and greater uv molar absorptivity and are more easily detected by subsequent mass spectrometry than the underivatized peptides.

Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H/D) exchange rates. The method has broad application to the study of protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.

Cooled sample introduction probe for liquid secondary ionization mass spectrometry.

The design and performance of a cooled sample introduction probe for fast atom bombardment or other liquid secondary ionization mass spectrometric studies are described. Cooling relatively volatile matrix materials (sulfolane, thioglycerol, and tetraglyme, for example) in situ in the ion source can increase the duration of the sample spectrum by a factor of 10. Cooling also permits a much wider range of matrix materials to be used. As an example, the spectrum of the carbohydrate, peracetyl [Glu(beta 1----3)]7 Glucitol in tetraglyme matrix, is shown to give an excellent spectrum including cleavage peaks corresponding to losses of the first five sugar residues. The spectrum lasted approximately 10 times longer when the probe tip was cooled to 9 +/- 1 degrees C than when no cooling was used, corresponding to a 10-fold increase in integrated sensitivity.

Artifacts in four-sector tandem mass spectrometry.

Several types of artifacts were shown to be present in 4-sector tandem collision-induced dissociation (CID) mass spectra. In CID spectra of protonated peptides produced by liquid secondary-ion mass spectrometry (LSIMS), peaks corresponding to successive losses of matrix molecules from the precursor ion were observed. In addition, peaks corresponding to MH+ ions of smaller peptides that were also present in the sample/matrix mixture in greater abundance than the selected precursor ion were observed. Both of these types of artifact peaks were shown to originate from the 'peak-at-every-mass' chemical noise at the same nominal mass as that selected by the first 2 sectors (MS1). These noise ions are transmitted through to the collision cell and produce fragments that are analysed and detected in the next 2 sectors (MS2). A second, unrelated, kind of artifact was found to be due to decompositions in the second field-free region of MS2 in an EBEB geometry machine. These artifacts, which are detectable over only a very limited mass range when using a conventional single-point detector, can be present over a much greater mass range when an array detector is used and when the collision cell is floated above ground potential. A clear understanding of the origins of all peaks in a CID spectrum is important in order to have a firm foundation for interpretation, manual or computer-aided, of the spectra of unknown compounds.

N-terminus determination: FAD and NADP binding domain mapping of hog liver flavin-containing monooxygenase by tandem mass spectrometry.

Highly purified hog liver flavin-containing monooxygenase was sequentially denatured, reduced, carboxymethylated, and digested with endoproteinase Glu-C. The purified peptides were subjected to mass spectrometric analysis and the amino acid sequence of selected fragments was determined by tandem mass spectrometry. The amino acid sequence of the first 12 residues of the N-terminus was: Ac-Ala-Lys-Arg-Val-Ala-Ile-Val-Gly-Ala-Gly-Val-Ser-Gly. The amino acid sequence determined for another peptide was: Lys-Ser-Val-Leu-Val-Val-Gly-Met-Gly-Asn-Ser-Gly-Thr-Asp-Ile-Ala-Val-Glu. The results provide direct evidence for the structure of the N-terminal modification of the protein and for the existence of the FAD and NADP binding domains of Gly-X-Gly-X-X-Gly.

Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry.

EcoRI DNA methyltransferase (MTase) is rapidly inactivated by N-ethylmaleimide with concomitant incorporation of 2 mol of N-ethyl[2-3H]maleimide/mol of functional monomer. Preincubation of the enzyme with either S-adenosylmethionine or DNA reduces the rate of activity loss, whereas preincubation with DNA and the S-adenosylmethionine analog sinefungin completely protects the enzyme from inactivation. An endo proteinase Glu-C digest of N-ethyl[2-3H]maleimide-modified enzyme was prepared and separated by high pressure liquid chromatography. Modified and unmodified cysteine-containing peptides were located and identified by radioactivity, mass spectrometry, and tandem mass spectrometry. In the absence of any ligands, cysteines 25, 116, and 223 are modified by N-ethylmaleimide; in the presence of DNA and sinefungin, Cys-223 is essentially unmodified. Thus, N-ethylmaleimide modification of Cys-223 in EcoRI DNA MTase is responsible for the loss of enzyme activity. Cys-223 is preceded by Asn, and this (or Cys-Asn) occurs with high frequency in adenine and cytosine (N-4) DNA MTases. Direct involvement of cysteine in methyl transfer reactions to adenine N-6 and cytosine N-4 is supported by the similarity of the reactions catalyzed by adenine N-6 and cytosine N-4 DNA MTases, the frequent presence of Asn-flanking Cys, and the importance of Cys-223 to EcoRI MTase function.

Analysis of hydrophobic proteins and peptides by electrospray ionization mass spectrometry.

During the last decade mass spectrometry has become an essential tool for the analysis of peptides and proteins. Electrospray ionization mass spectrometry (ESIMS) is one of several recently developed techniques for the determination of accurate molecular masses of proteins, peptides, and other biopolymers up to > 100 kDa. Up to the present, analyses have been performed mainly on biopolymers that are soluble in aqueous solutions. Mass spectrometric analyses of very hydrophobic species, such as membrane proteins, have seldom been reported in the literature. This is mainly due to the incompatibility between most mass spectrometric techniques and detergents and/or salts which are required to retain such proteins in solution. Hydrophobic proteins (for example, bacterioopsin) and peptides are in general not soluble in the solutions (methanol/water or acetonitrile/water) typically used for ESIMS, and most detergents and chaotropes interfere with the analysis. We have developed sample handling protocols and solvent systems that are compatible with instrumental requirements and also are capable of retaining very hydrophobic peptides and proteins in solution. Chloroform/methanol/water mixtures were found to work well with, e.g., bacterioopsin, and also to be compatible with samples dissolved in hexafluoroisopropanol and 70-95% formic acid.

Photoreactions of thymine and thymidine with N-acetyltyrosine.

We report here the photoinduced formation of a thymine-N-acetyltyrosine adduct. Irradiation of dilute solutions of thymine in the presence of N-acetyltyrosine (NAT) leads to the formation of N-acetyl-4-hydroxy-3-(6-hydrothymin-5-yl)phenylalanine (I), isolated as a mixture of the 5R and 5S diastereoisomers; the photoreaction occurs when irradiation is done either at lambda = 254 nm or at wavelengths of lambda > 290 nm. Irradiation of thymidine in the presence of NAT and of thymine in the presence of tyrosine leads to analogous photoadducts. The photoreaction of thymine with NAT is completely quenched by oxygen and cannot be sensitized by acetone. The likely mechanism involves initial photoionization of the amino acid and deprotonation to form the phenoxyl radical. Thymine then probably captures the released aqueous electron, leading to protonation at C6 of the resulting radical anion. Combination of the phenoxyl and 5,6-dihydrothymin-5-yl radicals would then lead to formation of the final products. The quantum yield for production of the thymine-NAT adduct at pH 7.8 was estimated to be about 5.5 x 10(-4), while a value of 2.3 x 10(-3) was estimated for production of corresponding thymidine adduct at pH 8.1. The dependence of the quantum yield for adduct formation on pH has been determined for both the thymine and thymidine reactions with NAT; the maxima in the quantum yield profiles occur at pH 8-8.5, while appreciable values were measured at pH 7.5. We have also demonstrated that a similar reaction occurs when tyrosine is located within a peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural evidence for an anion-directing track in the hen ovotransferrin N-lobe: implications for transferrin synergistic anion binding.

The transferrins are a class of iron-binding proteins that require the presence of a synergistic anion for conformation-dependent binding of ferric ions. Bromopyruvate, a known synergistic anion and affinity label of ovotransferrin (oTF) [Bailey, C. T., Patch, M. G., & Carrano, C. J. (1988) Biochemistry 27, 6276-6282], was used to probe the structure of the metal- and anion-binding sites of the functional N- and C-terminal proteolytic halves (oTF/2N and oTF/2C, respectively) of ovotransferrin. Incubation of oTF/2N with [2-14C]bromopyruvate in the presence of Fe3+ ions resulted in the incorporation of 0.70 mol of 14C label/mol of oTF/2N; 14C-labeled oTF/2N was then purified and digested sequentially with trypsin and V8 protease to determine the sites of modification. Quantification of 14C radioactivity, analysis of purified 14C-labeled peptides by gas-phase sequencing and mass spectrometry demonstrated that chemical modification was restricted to nucleophilic residues contained in a fragment corresponding to residues 189-204 of oTF/2N, including Lys 199, Lys 202, and His 196. Lysine 199 was also protected from modification with [3H]CH2O in iron-saturated oTF/2N, suggesting the involvement of this residue in anion binding by the apo conformation [Anderson, B. F., Baker, H. M., Norris, G. E., Rumball, S. V., & Baker, E. N. (1990) Nature 344, 784-787]. Lysine 199 is conserved as a basic residue in the N-terminal metal-binding domains of the transferrins but not in the homologous C-terminal metal-binding domains. Identical trials with oTF/2C showed binding, but not modification, with bromopyruvate. These data suggest that Lys 199, Lys 202 and His 196, which are located on an alpha-helix (8) that terminates at the anion-binding site [Dewan, J. C., Mikame, B. Hirose, M., & Sacchettini (1993) Biochemistry 32, 11963-11968], attract and channel the synergistic anion to the anion-binding site. The presence or absence of basic residues in the metal-binding lobes of transferrins may account for the different anion- and metal-binding characteristics observed for the iron-binding sites of these proteins.

Identification of protein-protein interfaces by decreased amide proton solvent accessibility.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein-protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5-24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5-24)-binding site. A complex of unknown structure also was analyzed, human alpha-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4-5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity.

Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins.

A combination of mass spectrometric (MS) techniques [electrospray MS, liquid secondary ion MS (LSIMS) and MS-MS] has been used for variant hemoglobin (Hb) detection and characterization. Electrospray MS allowed analysis of mixtures of intact globins giving simultaneously the molecular weights (accuracy 1-2 Da) and information about relative amounts of globins present. Currently, 14 Da is the minimum molecular weight difference required experimentally to accurately measure different species present in a mixture of 15-16 kDa proteins. Thus 80 and 79% of the known variants of alpha and beta chains, respectively, can be detected in mixtures with their normal counterparts, including Hb S (molecular weight difference = 30 Da). Abnormal hemoglobins detected were fractionated by C4 reversed-phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested by trypsin. The tryptic peptides were separated by C18 reversed-phase HPLC and analyzed by LSIMS to narrow down the mutation site to a single peptide. The mass measured in LSIMS frequently corresponded to a unique structure, thus giving the unequivocal identification of the mutation and its site. Where there was ambiguity, tandem MS on a Kratos Concept four-sector instrument was used for sequencing the abnormal peptide. The practical use of the methodologies presented is illustrated through analysis and identification of Hb G-San Jose, Hb Stanleyville II, Hb S and Hb Willamette.

Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.

The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.

Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5.

The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b5 were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from a Staphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariush et al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b5 were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2 +/- 2 and 9502.3 +/- 2 for bovine and rabbit trypsin-solubilized cytochrome b5, respectively. In both cases, these molecular weights correspond to a cytochrome b5 fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b5 was found to be 10,144.5 +/- 2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1-88) of Mr 10,146.1.

Tandem mass spectrometry in the clinical analysis of variant hemoglobins.

A combination of mass spectrometric techniques (electrospray mass spectrometry, liquid secondary-ion mass spectrometry (LSIMS), tandem mass spectrometry) has been used for variant hemoglobin detection and characterization. Electrospray mass spectrometry allowed analysis of mixtures of intact globins giving the molecular weights (accuracy 1-2 Da), and information about relative amounts of globins present, simultaneously. Abnormal hemoglobins detected in this way and by other means (screening, clinical symptoms) were fractionated by C-4 reverse phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested with trypsin. The tryptic peptides were separated by C-18 reverse phase HPLC and analysed by LSIMS to narrow down the mutation site to a single peptide. In some instances, the molecular weight of a variant peptide was sufficient to determine the mutation uniquely. When molecular weight information alone was insufficient to identify the mutation and its site, the peptide was sequenced by tandem mass spectrometry on a 4-sector instrument. In cases where more than one possible mutation site was present in the peptide and the mutation resulted in a change of only 1 Da in the peptide mass, the resolution and mass measurement accuracy of the 4-sector machine were essential in determining the correct sequence. The practical application of the methodologies presented is illustrated by the identification and analysis of Hb G-San Jose, Hb Willamette and D-Iran.

Evidence for complex formation between rabbit lung flavin-containing monooxygenase and calreticulin.

Rabbit lung flavin-containing monooxygenase (FMO, EC 1.14.13.8) was denatured, reduced, carboxymethylated, digested with endoproteinase Glu-C or trypsin, and subjected to mass spectrometric analysis. The amino acid sequences of selected peptides were determined by tandem mass spectrometry. Over 90% of rabbit lung FMO was mapped by liquid secondary ion mass spectrometry (LSIMS). The FMO N-terminal amino acid was found to be N-acetylated, and the N-terminal 23 amino acid peptide contained an FAD binding domain consisting of Gly-X-Gly-X-X-Gly. Another peptide was found to contain a NADP+ binding domain consisting of Gly-X-Gly-X-X-Ala. The mapped and/or sequenced peptides were found to be completely consistent with the peptide sequence deduced from the cDNA data and the previously published gas-phase sequencing data. Further mass spectrometry and protein analytical work unambiguously showed that rabbit lung FMO existed in tight association with a calcium-binding protein, calreticulin. Over 68% of rabbit lung calreticulin was mapped by LSIMS. Tandem mass spectrometric and gas-phase sequencing studies provided direct evidence for the identification of the N-terminal and other rabbit lung calreticulin-derived peptide sequences that were identical to other previously reported calreticulins. The complexation of calreticulin to rabbit lung FMO could account for some of the unusual physical properties of this FMO enzyme form.

Cumene hydroperoxide-mediated inactivation of cytochrome P450 2B1. Identification of an active site heme-modified peptide.

Cumene hydroperoxide (CuOOH)-mediated inactivation of cytochromes P450 (P450) results in the degradation of their prosthetic heme to products that alkylate the apoprotein. Indirect approaches suggest that this alkylation occurs at the active site. in order to identify the specific apoprotein site(s) alkylated, purified 3H- or 14C-heme-labeled P450 2B1 was incubated with CuOOH and subjected to lysyl endopeptidase-C digestion. Two major peaks (L1 and L2) containing 3H- or 14C-labeled peptides were detected by reverse-phase high pressure liquid chromatography of the digest. L1 contained the highest specific radioactivity and after Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded 3 peptide bands (M(r) approximately 3,500 (P1), 5,000 (P2), and 7,000 (P3)). Although all 3 bands were found radiolabeled, the yield of P1 was higher than that of P2 or P3. Amino acid sequence analysis of the first 13 N-terminal residues of P1 revealed the sequence RICLGEGIARNEL, corresponding to residues 434-446 of the reported 2B1 sequence. A species with the molecular mass of 3771 +/- 1 Da was detected in preliminary electrospray mass spectrometric analysis of L1. Since the theoretical average mass of the predicted peptide (residues 434-466) is 3721.99 Da, the additional 49 +/- 1 Da are considered to be contributed by the alkylating heme fragment. This alkylated 2B1 sequence contains not only Cys436, the conserved residue that provides the SH ligand for heme, but also other highly conserved residues, and therefore corresponds to the heme-sandwiching helix L of P450cam. To our knowledge, this is the first report to localize CuOOH-induced heme alkylation of 2B1 to its active site.