The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer.

A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.

Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit. Reevaluation of the side-by-side, antiparallel subunit model.

The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.

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.

Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4.

Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction of their prosthetic heme to reactive fragments that irreversibly bind to the protein. We have attempted to characterize this process structurally, using purified, 14C-heme labeled, recombinant human liver P450 3A4 as the target of CuOOH-mediated inactivation, and a battery of protein characterization approaches [chemical (CNBr) and proteolytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman sequencing, and mass spectrometric analyses]. The heme-peptide adducts isolated after CNBr/lysylendopeptidase-C digestion of the CuOOH-inactivated P450 3A4 pertain to two distinct P450 3A4 active site domains. One of the peptides isolated corresponds to the proximal helix L/Cys-region peptide 429-450 domain and the others to the K-region (peptide 359-386 domain). Although the precise residue(s) targeted remain to be identified, we have narrowed down the region of attack to within a 17 amino acid peptide (429-445) stretch of the 55-amino acid proximal helix L/Cys domain. Furthermore, although the exact structures of the heme-modifying fragments and the nature of the adduction remain to be established conclusively, the incremental masses of approximately 302 and 314 Da detected by electrospray mass spectrometric analyses of the heme-modified peptides are consistent with a dipyrrolic heme fragment comprised of either pyrrole ring A-D or B-C, a known soluble product of peroxidative heme degradation, as a modifying species.

N-Glycosylation of pig flavin-containing monooxygenase form 1: determination of the site of protein modification by mass spectrometry.

By using a combination of biochemical methods (i.e., endoglycosidase H digestion and immunoblot and plant lectin binding studies), it was verified that pig flavin-containing monooxygenase (FMO1) was N-glycosylated. By using mass spectrometry approaches [i.e., peptide mapping, gas chromatography/mass spectrometry, microbore HPLC/electrospray ionization mass spectrometry (LC/ESI/MS), chemical ionization gas chromatography/mass spectrometry (CI/GC/MS), and matrix-assisted laser desorption mass spectrometry (MALDI/MS)], we were able to confirm that pig FMO1 was N-glycosylated and we were able to identify the site of N-glycosylation. Pig FMO1 contains two putative consensus sites of N-glycosylation. The results showed that pig FMO1 amino acid Asn120 was selectively N-glycosylated. Highly purified pig FMO1 avidly bound concanavalin A and reacted positively for carbohydrates by the periodic acid/Schiff's base method of analysis. In addition, treatment of pig FMO1 with endo-N-acetylglucosaminidase converted the enzyme to another species with a molecular mass approximately 5000 Da lower than that of the parent protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments. Peptide mapping of pig FMO1 showed that the protein used in the study was not contaminated with another glycoprotein. MALDI/MS experiments showed that pig FMO1 was present with the expected molecular mass but that higher-molecular mass forms consistent with the presence of N-linked high-mannose oligosaccharide structures were also covalently attached to the enzyme. The presence of N-acetylglucosamine isolated from acid hydrolysates of the N-linked high-mannose oligosaccharide of pig FMO1 was confirmed by high-pH anion exchange HPLC studies and verified by CI/GC/MS studies of derivatized monosaccharide fractions. Further analysis of pig FMO1 proteolytic peptides by LC/ESI/MS showed that the only residue that was N-glycosylated in pig FMO1 was Asn120. Knowledge of the structural aspects of FMO may be useful in understanding the membrane association properties of the enzyme.

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.

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.

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.

Accurate mass measurements using MALDI-TOF with delayed extraction.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100-500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

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 the heme adduct and an active site peptide modified during mechanism-based inactivation of rat liver cytochrome P450 2B1 by secobarbital.

The olefinic barbiturate secobarbital (SB) is a sedative hypnotic known to be a relatively selective mechanism-based inactivator of rat liver cytochrome P450 2B1. Previous studies have demonstrated that such inactivation results in prosthetic heme destruction and irreversible drug-induced protein modification, events most likely triggered by P450 2B1-dependent oxidative activation of the olefinic pi-bond. However, the precise structure of the SB-modified heme and/or the protein site targeted for attack remained to be elucidated. We have now isolated the SB-heme adduct from P450 2B1 inactivated by [14C]SB in a functionally reconstituted system and structurally characterized it by electronic absorption spectroscopy and tandem collision-induced dissociation (CID), matrix-assisted laser desorption ionization on time of flight (MALDI-TOF), and liquid secondary ion mass spectrometry in the positive mode (+ LSIMS) as the N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX adduct. The [14C]SB-modified 2B1 protein has also been isolated from similar inactivation systems and subjected to lysyl endopeptidase C (Lys-C) digestion and HPLC-peptide mapping. A [14C]SB-modified 2B1 peptide was thus isolated, purified, electrotransferred onto a poly-(vinylidene) membrane, and identified by micro Edman degradation of its first N-terminal 17 residues (S277NH(H)TEFH(H)ENLMISLL293) as the Lys-C peptide domain comprised of amino acids 277-323. This peptide thus includes the peptide domain corresponding to the distal helix I of P450 101, a region highly conserved through evolution, and which is known not only to flank the heme moiety but also to intimately contact the substrates. This finding thus suggests that SB-induced protein modification of P450 2B1 also occurs at the active site and, together with heme N-alkylation, contributes to the SB-induced mechanism-based inactivation of P450 2B1.

There are three distinct forms of bombesin. Identification of [Leu13]bombesin, [Phe13]bombesin, and [Ser3,Arg10,Phe13]bombesin in the frog Bombina orientalis.

Amphibian bombesin is the prototypic peptide that defines the bombesin-like peptide family. In this paper we show that in the frog Bombina orientalis, there are actually 3 distinct forms of bombesin, and each of these peptides is an agonist with differing affinities for the known bombesin receptors. Oligonucleotides complementary to the 5'- and 3'-untranslated regions of the bombesin mRNA were used to amplify bombesin-related cDNAs from the skin, brain, and gut of B. orientalis. Three classes of cDNAs were found. One class encoded the previously characterized form of bombesin which has a Leu at position 13 ([Leu13]bombesin). The other two classes, respectively, encoded new bombesin-like peptides which we have designated as [Phe13]bombesin and [Ser3,Arg10,Phe13]bombesin ([SAP]bombesin). The existence of [SAP]bombesin in skin was confirmed by tandem mass spectrometry. Polymerase chain reaction analysis of genomic DNA showed the mRNAs for [Leu13]bombesin, [Phe13]bombesin, and [SAP]bombesin most likely arise from separate genes. Polymerase chain reaction analysis showed different patterns of tissue-specific expression for each form. [Leu13]Bombesin and [SAP]bombesin were predominantly expressed in skin, brain, and gut; [Phe13]bombesin was expressed only in brain, and [Leu13]bombesin predominated in oocytes. [SAP]Bombesin contained a cleavage site between residues 4 and 5, which if used would yield the peptide [SAP]bombesin(5-14) which has the sequence [Gln3,Arg6]neuromedin B. Thus a frog homolog of NMB could derive from the [SAP]bombesin prohormone. [Phe13]Bombesin, [SAP]bombesin, and [SAP]bombesin(5-14) were synthesized and their affinities for the mammalian bombesin-like peptide (GRP and NMB) receptors determined. These peptides acted as agonists for the GRP and NMB receptors, with relative potencies for the GRP receptor of [Leu13]bombesin > [Phe13]bombesin > [SAP]bombesin(5-14) > [SAP]bombesin and for the NMB receptor of [Phe13]bombesin > [SAP]bombesin(5-14) > [Leu13]bombesin > [SAP]bombesin. None of these peptides demonstrated high affinity binding for the BRS-3 receptor. The different receptor affinities and tissue distribution of these peptides suggests distinct physiologic roles and raises the possibility of as yet uncharacterized mammalian homologs of these new amphibian peptides.

Hemoglobin D Ibadan-beta zero thalassemia: detection by neonatal screening and confirmation by electrospray-ionization mass spectrometry.

We describe an infant with hemoglobin D Ibadan-beta zero thalassemia whose hemoglobinopathy was initially detected by neonatal screening. This previously undescribed condition was confirmed by family studies and by globin chain analysis by mass spectrometric techniques. The case illustrates the importance to neonatal screening programs of confirmatory testing and of linkage with reference laboratories capable of globin chain analysis. Hematologic studies at 36 months of age suggested that the presence of hemoglobin D Ibadan had no deleterious effect on this child with heterozygous beta zero thalassemia.

Mapping a membrane-associated conformation of colicin Ia.

Channel-forming colicins exist in at least two different membrane-associated conformations: a voltage-independent closed-channel state and a voltage-dependent open-channel state. In a voltage-independent membrane-associated conformation, we find that two major regions of colicin Ia are protected from pepsin proteolysis after association with negatively charged membranes. In contrast, colicin Ia is rapidly and completely proteolyzed in the absence of membranes. The major protected region includes an electrophysiologically defined C-terminal channel-forming domain as well as 96 residues upstream of this region. Approximately 100 residues spanning Ala79- approximately Arg189 within the N-terminal domain are protected as well. The first N-terminal 76 residues of colicin Ia and a large region which includes much of the putative central receptor-binding domain are not protected from proteolysis. Both N- and C-termini of protected peptides have been identified using a combination of gel electrophoresis, N-terminal sequencing, and mass spectrometry, thereby defining specific residues that are located on the outside of the lipid bilayer. These data suggest a role for regions other than the electrophysiologically defined C-terminal channel-forming domain in membrane insertion and channel formation.

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.

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.

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.

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)

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.

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.