Selective oxidation in vitro by myeloperoxidase of the N-terminal amine in apolipoprotein B-100.

In contrast to the multiple low abundance 2,4-dinitrophenylhydrazine-reactive tryptic peptides formed by oxidation of LDL with reagent HOCl in vitro, myeloperoxidase-catalyzed oxidation produces a dominant product in considerably greater yield and selectivity. This modified peptide had a single amino-terminal sequence corresponding to amino acids 53-66 of apolipoprotein B-100 (apoB-100), but its mass spectra indicated a significantly higher mass than could be reconciled with simple modifications of this peptide. Subsequent studies indicate that this product appears to result from N-chlorination of the N-terminal amino group of apoB-100 and dehydrohalogenation to the corresponding imine, which may form the hydrazone derivative directly, or after hydrolysis to the ketone. The methionine residue is oxidized to the corresponding sulfoxide, and the primary sequence peptide (residues 1-14 of apoB-100) is linked by the intramolecular disulfide bond between C-12 and C-61 to the peptide composed of residues 53-66, as we have observed previously (Yang, C-Y., T. W. Kim, S. A. Weng, B. Lee, M. Yang, and A. M. Gotto, Jr. 1990. Proc. Natl. Acad. Sci. USA. 87: 5523-5527) in unmodified LDL. The selective oxidation by myeloperoxidase of the N-terminal amine suggests strong steric effects in the approach of substrate to the enzyme catalytic site, an effect that may apply to other macromolecules and to cell surface molecules.

Automatic identification of proteins with a MALDI-quadrupole ion trap mass spectrometer.

A matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer of new design is described. The instrument is based on a commercial Finnegan LCQ ion trap mass spectrometer to which we have added a MALDI ion source that incorporates a sample stage constructed from a compact disk and a new ion transmission interface. The ion interface contains a quadrupole ion guide installed between the skimmer and the octapoles of the original instrument configuration, allowing for operation in both MALDI and electrospray ionization modes. The instrument has femtomole sensitivity for peptides and is capable of collecting a large number of MALDI MS and MALDI MS/MS spectra within a short period of time. The MALDI source produces reproducible signals for 10(4)-10(5) laser pulses, enabling us to collect MS/MS spectra from all the discernible singly charged ions detected in a MS peptide map. We describe the different modes of the instrument operation and algorithms for data processing as applied to challenging protein identification problems.

Rapidly switchable matrix-assisted laser desorption/ionization and electrospray quadrupole-time-of-flight mass spectrometry for protein identification.

We describe a new interface for a prototype quadrupole-quadrupole-time-of-flight (TOF) mass spectrometer (Centaur, Sciex) that allows rapid switching between electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) modes of operation. Instrument performance in both modes is comparable (i.e., resolution approximately 10,000 FWHM, mass accuracy <10 ppm, sensitivity approximately 1 fmol) because the ion source is decoupled from the TOF mass analyzer by extensive gas collisions in the quadrupole stages of the instrument. The capacity to obtain side-by-side high quality ESI and MALDI mass spectra from a single proteolytic mixture greatly facilitates the identification of proteins and elucidation of their primary structures. Improved strategies for protein identification result from this ability to measure spectra using both ionization modes in the same instrument and to perform MS/MS on singly charged as well as multiply charged ions. Examples are provided to demonstrate the utility and performance of the modified instrument.

Structural and functional characterizations of the proteasome-activating protein PA26 from Trypanosoma brucei.

The activated 20 S proteasome, which has been found only in mammalian cells, is composed of two heptamer rings of an activator protein on each end of the 20 S proteasome and is inducible by interferon-gamma. A 20 S proteasome has been recently identified in a protozoan pathogen Trypanosoma brucei, but there has been no experimental evidence yet for the presence of a 26 S proteasome. Instead, an activated form of 20 S proteasome was isolated from this organism, which has significantly enhanced peptidase activities. It consists of an additional activator protein with an estimated molecular mass of 26 kDa (PA26) (To, W. Y., and Wang, C. C. (1997) FEBS Lett. 404, 253-262). The profile and sequences of tryptic peptides from PA26 were determined by mass spectrometry; no matches were found in the data base. The peptide sequences were used in reverse transcriptase-polymerase chain reaction to isolate a full-length cDNA clone encoding PA26. The protein sequence thus derived from it indicates little sequence identity with those of mammalian activator proteins PA28 alpha, beta, or gamma. There is only a single copy of PA26 gene in T. brucei. Purified recombinant PA26 polymerizes spontaneously to form heptamer ring with an outer diameter of 8.5 nm. The ring binds and activates 20 S proteasomes from T. brucei as well as rat, whereas human PA28alpha can neither bind nor activate T. brucei 20 S proteasome. The former is thus apparently more ubiquitous than PA28 in its capability of binding to and activating 20 S proteasomes. Its presence in T. brucei may also suggest a more ancient origin of proteasome activator proteins and a much wider involvement in protein degradation among other eukaryotic organisms than was originally envisaged.

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties.

Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.

Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers.

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.

Sensitive high-resolution analysis of biological molecules by capillary zone electrophoresis coupled with reflecting time-of-flight mass spectrometry.

Off-line and on-line capillary zone electrophoresis-electrospray ionization time-of-flight mass spectrometry (CZE-ESI-TOF-MS) experiments were conducted using uncoated fused-silica capillaries coupled to a reflecting TOF mass spectrometer via a gold-coated sheathless interface. Off-line and on-line experiments were performed on standard mixtures of proteins and peptides. Samples collected off-line electrokinetically in plastic vials were analyzed by standard ESI-TOF-MS at the pmol level. Sheathless CZE-ESI-TOF-MS was first simulated in an off-line experiment, using a test bench, in order to select a suitable running electrolyte, to find the optimal electrospray potential, and also to test the gold-coated capillary tips. This enabled an ease of transition to on-line measurements. On-line CZE-ESI-TOF-MS measurements of the total ion electropherogram (TIE) and of selected ion electropherograms (SIE) on peptide mixtures demonstrated fmol-level sensitivity, with S/N values of 250-400 on raw data (TIE mode) and of 30-760 (SIE mode). The use of reflecting TOF-MS afforded mass resolution values R>6000 (m/delta(m)(FVHM)) and enabled isotopic resolution of peptide components as well as mass accuracy in the 10 ppm range. These results were comparable with values observed with the usual ESI source on the same mass spectrometer, and thus demonstrated no loss in spectral quality from using the sheathless CE interface. On-line CE separation efficiency was equivalent to that obtained off-line for the separation of a peptide mixture, with N=35000-87000 theoretical plates. Separations of standard proteins yielded equivalent mass spectral resolution and accuracy with separation efficiencies of N=2800-5500 and S/N values of 110-225 on raw data. The gold-coated sheathless CE-ESI interface was found to be relatively easy to prepare with the use of gold vapour deposition. The interface produced a stable electrospray for extended periods of time and was found to be robust and reliable.

Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry.

A mass spectrometer coupling electrospray ionization with time-of-flight mass spectrometry (ESI-TOFMS) has been used to investigate the oligomeric species of Escherichia coli citrate synthase, and to determine the effect of nicotinamide adenine dinucleotide (NADH), an allosteric inhibitor of this enzyme, on the equilibrium between the oligomeric forms. An equilibrium mixture of dimers (M = 95,770 Da) and hexamers (M = 287,310 Da) was found under the conditions used (KA = 6.9 x 10(10) M-2), and NADH was observed to bind selectively to the hexamer (KD = 1.1 microM), shifting the equilibrium to the latter form. The power of ESI-TOFMS to measure ions of very large mass-to-charge ratio (up to m/z approximately 10,000 in this case) is shown to be a valuable tool for obtaining accurate information about compositions of noncovalent complexes and equilibrium constants. The measured constants agree with those determined by conventional means.

Matrix-assisted laser desorption ionization of neutral clusters composed of matrix and analyte molecules.

A new version of the matrix-assisted laser desorption ionization experiment is described. Composed clusters created by laser desorption of a mixture of analyte and matrix material were injected into a supersonic jet and then analyzed by multiphoton ionization time-of-flight mass spectrometry. Selective two-photon ionization of matrix molecules of a cluster component caused their dissociation, followed by intercluster proton or cation transfer to an analyte that is transparent for the wavelength of ionizing radiation. The intercluster charge transfer reactions are the only pathway for analyte ion formation under these conditions, as can be concluded from analysis of the mass spectra of a variety of substances.