Search

Here we show that fragment ion abundances from dissociation of ions created from mixtures of multiply modified histone H4 (11 kDa) or of N-terminal synthetic peptides (2 kDa) correspond to their respective intact ion abundances measured by Fourier transform mass spectrometry. Isomeric mixtures of modified forms of the same protein are resolved and quantitated with a precision of [less than or equal to] 5% using the relative ratios of their fragment ions, with intact protein ions created by electrospray greatly easing many of the systematic biases that more strongly affect small peptides (e.g., differences in ionization efficiency and ion m/z values). The ion fragmentation methods validated here are directly extensible to intact human proteins to derive quantitative information on the highly related and often isomeric protein forms created by combinatorial arrays of posttranslational modifications.

The heterogeneous overexpression and purification of RapL and its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis are reported. The inhibitory properties of several compounds are characterized, which were previously proposed to interrupt RapL function in precursor-directed biosynthetic experiments.

The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and [sup.14]N/[sup.15]N metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the [sup.14]N/[sup.15]N metabolic labeling strategy, we achieve 98% [sup.15]N incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically grown control ([sup.14]N) cells. We devise quantitative methods for top-down analyses, including a correction factor for accurate protein ratio determination based upon the signal-to-noise ratio. Using a database of 200 yeast protein forms identified previously by top-down MS, we verify the intact mass tag concept for protein identification without tandem MS. Overall, we find that top-down MS promises work flows capable of large-scale proteome profiling using stable isotope labeling and the determination of > 5 protein ratios per spectrum.

Streamlined methods using proteolysis and reporter-coenzyme A analogues of four types are employed to enable the selective detection of peptides containing active site serine. Each active site peptide is quickly detected using Fourier transform mass spectrometry and further knowledge about digestion heterogeity and side reactions due to a mass shift signature on each active site peptide are gained.

The antifungal natural product pyoluteorin contains a 4, 5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PItL and enzymatically desaturated to the pyrrolyl-S-PItL. Surprisingly, the FAD[H.sub.2]-dependent halogenase PItA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PItL intermediate and then the 4,5-dichloropyrrolyl-S-PItL product. PItA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FAD[H.sub.2]. Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PItL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCI intermediate for capture by the electron-rich [C.sub.4] and [C.sub.5] of the heteroaromatic pyrrole moiety. electrospray ionization-Fourier transform MS | clorobiocin | coumermycin | flavin reductase | pyrrole biosynthesis

For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a 'top down' strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da [DELTA]m on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between [Asp.sup.90] and [Asp.sup.192], consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).

For localization of deuterium atoms after solution-phase exchange with [D.sub.2]O, intact proteins are often digested prior to analysis by mass spectrometry (MS) and tandem MS (MS/MS). Amelioration of limitations associated with this approach (e.g.,

The iron-containing heterodimeric MbnBC enzyme complex plays a central role in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, posttranslationally modified natural products that bind copper with high affinity. MbnBC catalyzes a four-electron oxidation of a cysteine residue in its precursor-peptide substrate, MbnA, to an oxazolone ring and an adjacent thioamide group. Initial studies of MbnBC indicated the presence of both diiron and triiron species, complicating identification of the catalytically active species. Here, we present evidence through activity assays combined with electron paramagnetic resonance (EPR) and Mossbauer spectroscopic analysis that the active species is a mixed-valent, antiferromagnetically coupled Fe(II)Fe(III) center. Consistent with this assignment, heterologous expression of the MbnBC complex in culture medium containing less iron yielded purified protein with less bound iron but greater activity in vitro. The maximally activated MbnBC prepared in this manner could modify both cysteine residues in MbnA, in contrast to prior findings that only the first cysteine could be processed. Site-directed mutagenesis and multiple crystal structures clearly identify the two essential Fe ions in the active cluster as well as the location of the previously detected third Fe site. Moreover, structural modeling indicates a role for MbnC in recognition of the MbnA leader peptide. These results add a biosynthetic oxidative rearrangement reaction to the repertoire of nonheme diiron enzymes and provide a foundation for elucidating the MbnBC mechanism. [ABSTRACT FROM AUTHOR]

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl suffate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within ~5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of ~400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of ~19 000 protein forms. This ALS-PAGE/ RPLC approach to proteome processing ameliorates the 'front end' problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.