Simon J. Gaskell

Metastable decomposition of peptide [M + H]+ ions via rearrangement involving loss of the C-terminal amino acid residue

A novel fragmentation of metastable peptide [M + H]+ ions is described. Loss of the C-terminal amino acid residue is accomqanied by retention of one of the carboxyl oxygens, as judged by 18O-labeling. The retained 8O label is located at the new C-terminus. Sequential mass spectrometric analyses indicate that the structure of the first-generation product ion is indistinguishable from that of the [M + H]+ ion of the peptide with one fewer amino acid residues. Thus, for example, the metastable decompositions of ions of m/z 904 are similar whether they correspond to des-Arg9-bradykinin [M + H]+ ions or to fragments derived from bradykinin [M + H]+ ions. No corresponding rearrangements have been observed for peptides with C-terminal amide or ester functions. The mechanism of this fragmentation may be considered to be analogous to that previously suggested for fragmentations of [M + alkali metal cation]+ ions. For the examples of bradykinin and related peptides, the rearrangement is strongly promoted when arginine is the amino acid residue lost. The same fragmentation is also favored by the presence of an arginine residue at or near the N-terminus. The strong influence of peptide amino acid composition, including residues remote from the C-terminus, on the prevalence of this fragmentation suggests mechanistic complexities that require further elucidation.

Influence of cysteine to cysteic acid oxidation on the collision-activated decomposition of protonated peptides: evidence for intraionic interactions

Oxidation of cysteine residues to cysteic acids in C-terminal arginine-eontaining peptides (such as those derived by tryptic digestion of proteins) strongly promotes the formation of multiple members of the Y− series of fragment ions following low energy collision-activated decomposition (CAD) of the protonated peptides, Removal of the arginine residue abolishes the effect, which is also attenuated by conversion of the arginine to dimethylpyrim-idylornithine. The data indicate the importance of an intraionic interaction between the cysteic acid and arginine side-chains. Low energy CAD of peptides which include cysteic acid and histidine residues, also provides evidence for intraionic interactions. It is proposed that these findings are consistent with the general hypothesis that an increased heterogeneity (with respect to location of charge) of the protonated peptide precursor ion population is beneficial to the generation of a high yield of product ions via several charge-directed, low energy fragmentation pathways. Furthermore, these data emphasize the significance of gas-phase conformations of protonated peptides in determining fragmentation pathways.

Sequential mass spectrometry applied to the study of the formation of “internal” fragment ions of protonated peptides

Abstract “Internal” fragment ions of protonated peptides arise by charge retention on a portion of the structure excised from the peptide chain. Their formation appears to be favored by the extented time scale and multiple collision conditions associated with decomposition experiments which use the r.f.-only quadrupole of a hybrid sector/quadrupole mass spectrometer. Sequential product ion scanning and reaction intermediate scanning techniques of MS—MS—MS were used to probe these necessarily multistep fragmentations. With an instrument of BEqQ geometry, mass-analyzed ion kinetic energy spectrometry analyses were used to determine the population of candidate intermediates available for subsequent decomposition to internal fragments in the r.f.-only quadrupole. Reaction intermediate scanning then indicates the relative quantitative significance of competing pathways. In the examples studied, certain Y″-type ions show a high inherent tendency to fragment further to give internal fragments, consistent with the formal equivalence of these intermediates to protonated truncated peptides. Internal fragments can also arise, however, through a number of multistep pathways which can predominate in situations where the requisite intermediates are formed in high abundance.

Dehydration of peptide [M + H]+ ions in the gas phase

The loss of water from protonated peptides was studied using [18O]-labeling of the C-terminal carboxyl group. The structures (including the location of the isotopic label) of first-generation product ions were examined by sequential product ion scanning (MS3 and MS4) using a hybrid sector/quadrupole mass spectrometer. Water loss may involve carboxylic acid groups, side-chain hydroxyls, or peptide backbone oxygens. Although one of these three pathways often predominates, more than one dehydration route can be operative for a single peptide structure. When peptide backbone oxygen is lost, the dehydration can occur at one or two primary sites along the backbone, with the location of the site(s) varying among peptides. When water loss involves the C-terminal carboxyl group, the resulting ion may undergo extensive intraionic oxygen isotope exchange. This evidence for complex intraionic interactions further emphasizes the significance of gas-phase conformation in determining the fragmentations of peptide ions.

Quantitation of leukotriene B4 in human serum by negative ion gas chromatography-mass spectrometry

Leukotriene B4 (LTB4) is a potent chemotactic agent formed via the 5-lipoxygenase pathway from arachidonic acid. To understand the role LTB4 plays in several pathological processes it is essential that endogenous concentrations of LTB4 be accurately quantitated. We have developed a method based on electron capture negative ion mass spectrometry for the analysis of LTB4 in serum at low picogram per milliliter concentrations. Blood is collected into the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) to suppress ex vivo formation. Serum is isolated, equilibrated with the internal standard [2H4]LTB4, and extracted using octadecyl-silica (C-18) cartridges. After conversion of the carboxylic acids to their pentafluorobenzyl esters the extract is purified by straight-phase HPLC. Gas chromatographic-mass spectrometric analysis is accomplished on the tert-butyldimethylsilyl ether derivatives using dual-selected ion monitoring of m/z 431 and 435. These ions correspond to loss of tert-butyldimethylsilanol from the (M-PFB)- ion of endogenous and [2H4]LTB4, respectively. The concentration of LTB4 in human serum samples was 10.0 +/- 4.0 pg/ml (n = 5). The assay exhibited satisfactory precision, with an intraassay coefficient of variation of 17% and a high degree of accuracy. The concentration of LTB4 in serum collected with (NDGA) was less than 10% of that observed in blood collected without the lipoxygenase inhibitor. Ex vivo formation can therefore be a major obstacle in assessing circulating levels of LTB4.

Multiple scan modes in the hybrid tandem mass spectrometric screening and characterization of the glutathione conjugate of 2-furamide.

The glutathione conjugate of 2-furamide has been screened for and structurally characterized by tandem mass spectrometry (MS(MS) by using a hybrid instrument of BEqQ design. Mass spectrometry experiments employed fast atom bombardment (FAB) ionization of a crude bile extract from a rat dosed with a 1:1 mixture of unlabeled and [ 13C12-furamide. Initial screening for glutathione conjugates employed constant neutral loss scanning to detect the loss of 129 u, corresponding to the loss of the γ-glutamyl moiety of the conjugates. By direct comparison with control bile, [M + H] + ions of m/z 417 and 418 were readily identified as candidate ions corresponding to the glutathione conjugates of unlabeled and 13C-labeled 2-furamide. Complementary screening information was generated by using a methylated bile extract, with constant neutral loss scanning to detect the loss of the methylated γ-glutamyl moiety (143 u). An alternative screening procedure employing parent ion scanning to detect the sodium adducts of methylated glutathione conjugates was also developed. Structural information was generated by frrst-generation product ion scanning of the protonated and sodium cationized forms of the candidate species, both native and derivatized. This provided a body of internally consistent evidence that the conjugate retains the pseudoaromatic furan ring system without ring hydroxylation. The utility of sequential mass spectrometry (MS(MS(MS) capability of the hybrid instrument in the analysis of complex biological mixtures was also demonstrated. Using the bile extract, first-generation product ions that formed in either the first or second field-free region of the double-focusing portion of the instrument were subsequently collisionally activated in the rf-only quadrupole followed by mass analysis of the second-generation product ions. Structural information so provided for the glutathione conjugate of 2-furamide further substantiated its retention of the pseudoaromatic furan ring system and facilitated plausible assignment of structures to ionic species generated through multiple decomposition events.

Decompositions of cationized heterodimers of amino acids in relation to charge location in peptide ions

The unimolecular decompositions of protonated heterodimers of native and derivatized amino acids to yield the protonated monomers were studied as a guide to charge location in peptide ions. Analyses using a hybrid instrument of BEqQ geometry demonstrated the advantages (with respect to mass resolution, sensitivityr reproducibility, and the elimination of extraneous signals) of the detection of product ions formed in the radiofrequency-only quadrupole region (q) rather than in the field-free region between Band E. Conversion of arginine to dimethylpyrimidylomithine (DMPO) reduced the proton affinity, as evidenced by the decomposition of the protonated arginine/DMPO heterodimer. Conversion of cysteine to pyridylethylcysteine enhanced the proton affinity. Application of these derivatization procedures to peptides resulted in changes in the observed fragmentations of the protonated precursors consistent with the predicted modifications in charge location. Unimolecular decomposition of the protonated dimer composed of glycine and N-acetylglycine yielded both protonated monomers with abundances differing by a factor of only 2; this suggests that in protonated peptides, the amide bonds are competitive with the N-terminal amino group as sites of protonation. It is clear that the propensities to proton’ or metal-cation location at particular sites in peptides are influenced by both short- and long-range intraionic interactions. In peptides composed of amino acids of similar cation affinities, it may be postulated that the ion population is heterogeneous with respect to the site of charge, with consequent promotion of multiple low-energy fragmentation routes.

Tandem mass spectrometric characterization of a specific cysteic acid residue in oxidized human apoprotein B-100.

The oxidation of low density lipoprotein (LDL) in vivo may result in its unregulated uptake by macrophages, with the consequent accumulation of cholesterol that is characteristic of the development of atherosclerosis. This paper describes initial experiments to elucidate structural changes that occur in an in vitro model of LDL oxidation. LDL was isolated from human blood and oxidized in the presence of copper ion. Lipid was removed and the isolated apoprotein was subjected to tryptic hydrolysis. The hydrolysate was separated by high performance liquid chromatography and individual fractions were screened by amino acid analysis to detect cysteic acid residues. Appropriate fractions were analyzed by fast atom bombardment mass spectrometry and hybrid tandem mass spectrometry. In this manner a tryptic fragment was identified that corresponded to residues 4187-4195 (EELCTMFIR), in which the cysteine and methionine residues were oxidized to cysteic acid and methionine sulfoxide, respectively. Identical analysis of LDL not subjected to in vitro oxidation revealed no evidence for this oxidized peptide. Earlier work established a surface location for this cysteine residue (Cys24) on the LDL particle, which suggested that its modification may significantly affect the properties of LDL, such as the propensity to intermolecular interaction via disulfide bridges. The analytical protocol developed here (involving proteolysis, screening of peptide fragments, and tandem mass spectrometry analysis) constitutes a strategy of general applicability to the characterization of targeted modifications of large proteins via mass spectrometry.

Quantification of steroid conjugates using fast atom bombardment mass spectrometry

Fast atom bombardment/mass spectrometry or liquid secondary ion mass spectrometry provides the capability for direct analysis of steroid conjugates (sulfates, glucuronides) without prior hydrolysis or derivatization. During the analysis of biologic extracts, limitations on the sensitivity of detection arise from the presence of co-extracted material which may suppress or obscure the analyte signal. A procedure is described for the quantitative determination of dehydroepiandrosterone sulfate in serum which achieved selective isolation of the analyte using immunoadsorption extraction and highly specific detection using tandem mass spectrometry. A stable isotope-labeled analog [( 2H2]dehydroepiandrosterone sulfate) was used as internal standard. Fast atom bombardment of dehydroepiandrosterone sulfate yielded abundant [M-H]- ions that fragmented following collisional activation to give HSO4-; m/z 97. During fast atom bombardment/tandem mass spectrometry of serum extracts, a scan of precursor ions fragmenting to give m/z 97 detected dehydroepiandrosterone sulfate and the [2H2]-labeled analog with a selectivity markedly superior to that observed using conventional mass spectrometry detection. Satisfactory agreement was observed between quantitative data obtained in this way and data obtained by gas chromatography/mass spectrometry of the heptafluorobutyrates of dehydroepiandrosterone sulfate and [2H2]dehydroepiandrosterone sulfate obtained by direct derivatization.

Determination of high-energy fragmentation of protonated peptides using a BEqQ hybrid mass spectrometer

A hybrid tandem instrument of BEqQ geometry was used to determine high-energy decomposition of protonated peptides, such as side-chain fragmentation yielding dn and wn ions. The transmission through both E and Q of such product ions, formed in the second field-free region, permits improved mass resolution and confident mass assignment. The experimental technique may involve synchronous scanning of E and Q, or, for the purpose of identification of specific products, limited-range scanning of either E or Q with the other analyzer fixed. These techniques are not equivalent, with respect to product ion transmission, to the double focusing of product ions achieved with four-sector instruments but nevertheless represent a critical improvement over conventional mass-analyzed ion kinetic energy spectrometry analyses. Fragmentation of protonated peptides occurring in the second field-free region inside and outside the collision cell were distinguished by floating the collision cell above ground potential. Mass filtering using Q confirmed the mass assignments. The data indicate that product ions resulting from spontaneous decomposition are in some instances quantitatively more significant than those resulting from high-energy collisional activation. Furthermore, the differentiation of the products of low- and high-energy processes should facilitate spectral interpretation.