Klaus Biemann

Collision-induced fragmentation of (M + H)+ ions of peptides. Side chain specific sequence ions

Abstract The collision (10 keV)-induced decomposition (CID) of (M + H)+ ions of peptides generated by fast atom bombardment ionization has been studied in a tandem mass spectrometer consisting of two consecutive double-focussing analyzers. Two novel fragmentation processes are described; one (termed dn) leads to the formation of N-terminal ions that permit the differentiation of leucine isoleucine; the other leads to a new set of C-terminal ions (termed vn) and is related to the structure of the amino acid representing the N-terminus of the fragment. The mechanisms of formation are supported by B2/E and B/E scans, which define the precursor and product ions. These and other fragmentations of (M + H)+ ions under CID conditions and kilovolt collision energies seem to involve fragmentation at a site remote from the charge. The fragmentation processes which (M + H)+ ions of peptides undergo are related to the site of protonation and the degree to which the positive charge is fixed at that site.

Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin

BACKGROUND Angiogenesis, the formation of new blood vessels, is essential for tumor growth. The inhibition of angiogenesis is therefore emerging as a promising therapy for cancer. Two natural products, fumagillin and ovalicin, were discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. An analog of fumagillin, AGM-1470, is currently undergoing clinical trials for the treatment of a variety of cancers. The underlying molecular mechanism of the inhibition of angiogenesis by these natural drugs has remained unknown. RESULTS Both AGM-1470 and ovalicin bind to a common bifunctional protein, identified by mass spectrometry as the type 2 methionine aminopeptidase (MetAP2). This protein also acts as an inhibitor of eukaryotic initiation factor 2alpha (elF-2alpha) phosphorylation. Both drugs potently inhibit the methionine aminopeptidase activity of MetAP2 without affecting its ability to block elF-2alpha phosphorylation. There are two types of methionine aminopeptidase found in eukaryotes, but only the type 2 enzyme is inhibited by the drugs. A series of analogs of fumagillin and ovalicin were synthesized and their potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined. A significant correlation was found between the two activities. CONCLUSIONS The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin. This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs.

Reconstructed Mass Spectra, A Novel Approach for the Utilization of Gas Chromatograph—Mass Spectrometer Data

Abstract Peak profile analysis of each of the few hundred mass chromatograms generated during a complete GC-MS experiment identifies all m/e values which maximize at any of the few hundred consecutive mass spectra recorded during the gas chromatogram. The resulting sets of data correspond to the mass spectrum of each eluting component (even very minor ones) practically free of the contributions of unresolved companion substances, tailing fractions, column bleed, etc. These “reconstructed mass spectra” are therefore more easily interpreted or automatically identified than the mass spectra originally recorded. A plot of the summed abundance of the ions that maximize at a given spectrum index number (“mass resolved gas chromatogram”) results in a gas chromatogram of dramatically improved apparent resolution.

Matrix-assisted laser desorption ionization mass spectrometry with 2-(4-hydroxyphenylazo)benzoic acid matrix

A novel matrix substance, 2-(4-hydroxyphenylazo) benzoic acid, or HABA, has been found to be very advantageous for matrix-assisted ultraviolet laser desorption ionization mass spectrometry. This compound has been successfully used for the desorption of peptides, proteins, and glycoproteins up to approximately 250 kDa. For these materials, the most abundant analyte-related peaks correspond to [M + H]+ ions and multiply protonated molecules. Comparisons with sinapic acid, 2,5-dihydroxybenzoic acid, and α-cyano-4-hydroxycinnamic acid indicate that the new matrix provides comparable sensitivity for peptides and smaller proteins but results in better sensitivity for larger proteins and glycoproteins in protein mixtures. Other matrices discriminate against the higher mass components in these cases. Somewhat reduced mass resolution has been found for smaller proteins, but for larger proteins and glycoproteins the best mass resolution can often be obtained with the new matrix. For other classes of compounds that form ions predominantly via cation attachment, at least as good sensitivity and even better resolution have been obtained. Derivatized glycolipids and synthetic polymers have been studied in detail. For the analysis of many synthetic polymers, the best performance in terms of sensitivity and mass resolution has been observed with HABA matrix. Mass resolution was higher for cation adducts than for the protonated peptide molecules in the same mass range. The new matrix exhibits greatly extended (in time) analyte ion production and reproducibility. Owing to the uniform sample surface with this matrix, barely any spatial variation of the ion signal could be observed. In addition, many hundreds of single-shot mass spectra could be accumulated from the same spot, even for larger proteins.

UTILITY OF NON-COVALENT COMPLEXES IN THE MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY OF HEPARIN-DERIVED OLIGOSACCHARIDES

Molecular weights of heparin-derived oligosaccharides ranging from disaccharides to hexadecasaccharides have been determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. While these compounds ionize poorly or not at all when used as such, a strong signal can be obtained of their ionic complexes formed with a basic peptide or protein. The molecular weight of the sulfated oligosaccharide is determined by subtracting the mass of the basic component from that of the complex. Optimization of the experimental conditions resulted in sub-picomole sensitivity, in the elimination of sulfate loss and of the interference from attachment of inorganic cations. Synthetic peptides (Arg-Gly)10 and (Arg-Gly)15 were specifically designed as complexing agents for synthetic and natural heparin fragments up to decasaccharides. Accurate molecular weight determination on chemically homogeneous oligosaccharides (+/- 0.05%) unambiguously identified the number of saccharide units, and the number of O,N-sulfate and N-acetyl groups. For oligosaccharides larger than decasaccharides, a small basic protein, angiogenin (M(r) = 14,120), was used to form the complex (an inhomogeneous hexadecasaccharide fraction was the largest available for this study). For inhomogeneous samples larger than decasaccharides, the mass accuracy is lower (+/- 0.2-0.3%) but still suffices to determine the number of saccharide units present and to estimate the number of sulfate groups, except it is no longer possible to differentiate one sulfate from two N-acetyl groups (delta = 4 Da). However, taking into account known regularities of sulfation and acetylation, the specificity of heparin lyases and chemical degradation steps, the method promises to contribute significantly to the determination of the primary structure of heparin and other sulfated glycosaminoglycans.

Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells.

BACKGROUND The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however. RESULTS Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation. CONCLUSIONS Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.

A comparison of keV atom bombardment mass spectra of peptides obtained with a two-sector mass spectrometer with those from a four-sector tandem mass spectrometer

Abstract The information content of the conventional fast (keV) atom bombardment (FAB) mass spectra of six peptides, substance P, des-Arg1substance P. Fibrinopeptide A, des-Arg16fibrinopeptide A, oxidized somatostatin, and reduced somatostatin, ranging in molecular weight from 1190.6 to 1638.7 is compared with that of the corresponding spectra measured by a high-performance (four-sector) tandem mass spectrometer after collision-induced decomposition (CID). The CID spectra exhibit distinct peaks corresponding to extended series of sequence characteristic ions free of the chemical background and contribution of the matrix which makes the weak fragment ion signals in conventional FAB mass spectra so difficult to interpret. The presence of arginine, a strongly basic amino acid, in either the N-terminal or C-terminal position is reflected in the dominance of characteristic N-terminal and C-terminal fragment ions. The resolving power of the first of the two mass spectrometers is clearly sufficient to permit the recording of separate CID spectra of oxidized and reduced somatostatin in a mixture.

Microderivatization of peptides by placing a fixed positive charge at the N-terminus to modify high energy collision fragmentation

Abstract For the differentiation of leucine and isoleucine in a peptide by high energy collision-induced dissociation (CID), it is generally required that there is a basic amino acid present at or near either the C-terminus or the N-terminus of the peptide. In these cases, fragmentation or the β,γ bond of the side chain occurs, generating ions designated w n or d n that permit the differentiation of these isomeric amino acids. While trypsin and Endo Lys-C generate peptides with a basic C-terminal amino acid, other enzymes cleave at neutral or acidic amino acids and may thus produce proteolytic peptides that do not contain any basic amino acids. For these, a microderivatization method has been developed that places a fixed positive charge at the C-terminus. It involves exposure of the peptide(s) deposited on the inner wall of a capillary tube, first to chloroacetyl chloride vapor and then to trimethylamine and water vapors. This two-step reaction attaches a trimethylammonium acetyl (TMA) moiety to the N-terminal amino group of the peptide. The CID spectra of these derivatives are very simple, exhibit the same characteristics (including abundant d n ions) as peptides bearing the strongly basic arginine at the N-terminus, and thus permit the differentiation of leucine from isoleucine. The reaction can be carried out at the sub-nanomole level.

Design considerations, calibration and applications of an array detector for a four-sector tandem mass spectrometer

Abstract A focal plane detector consisting of a channel electron multiplier array, phosphor-coated fiber optics and photodiode array has been constructed and installed at the final detector of a four-sector tandem mass spectrometer. The active length of the detector is 2 in., incorporating 2048 photodiodes and covering 6.6% of mass range. To obtain a complete tandem mass spectrum, a number of consecutive segments are recorded and assembled. Data acquisition and processing are carried out by a dedicated processor and a VAX 750 computer, respectively. Mass and gain calibration of the detector is carried out by using CsI ionized by fast atom bombardment in the first mass spectrometer. Experiments with two peptides, renin substrate ( M r 1757.9) and ACTH (18–39) ( M r 2464.2), gave excellent product ion spectra when 10 and 50 pmol were used, respectively. This represents at least a 50-fold decrease in sample requirement (compared with conventional detectors), whereas the quality of the spectra and mass assignments are equal to those of conventional detectors, or better. The detector functions equally well in the negative ion mode, as demonstrated with a ganglioside (GM 1 ) of M r 1545.9. All elements of the array detector, including the diode array, are mounted within the vacuum envelope of the mass spectrometer. It can be adjusted in angle and position through a bellows arrangement for focusing or adaptation to other modes of operation in which the focal plane of the spectrometer changes angle or position.

[4] Identification of posttranslationally modified amino acids in proteins by mass spectrometry

Publisher Summary This chapter illustrates strategies and methods for identification of posttranslationally modified amino acids in proteins by mass spectrometry. Mass spectrometry (MS) is particularly well suited for the investigation of compounds whose structure is unknown and for which reference compounds do not exist. The particular MS method employed is dictated by the level of information desired as well as the particular instrumentation available. Mass spectrometric protein sequencing strategies is divided into two broad categories—namely, (1) those which employ conventional ionization methods, such as electron impact (EI) or chemical ionization (CI), and require that the amino acid or polypeptide be chemically derivatized and (2) those which are capable of directly analyzing underivatized large or polar molecules–the soft ionization methods. The soft ionization methods eliminate the need for prior chemical derivatization which may destroy sensitive functional groups, requires more material and is often tedious. Fast atom bombardment mass spectrometry allows tackling much larger molecules such as peptides up to 10,000 daltons.