Biomedical applications of high-performance liquid chromatography—mass spectrometry with continuous-flow fast atom bombardment

This report describes the application of high-performance liquid chromatography combined with continuous-flow fast atom bombardment mass spectrometry to analytical problems in the biomedical laboratory. Applications include the compound-specific detection of diagnostic acylcarnitines in human urine, the separation and analysis of acyl-coenzyme A thioesters, and qualitative studies on complex mixtures of modified peptides (dansyl and dinitrophenyl derivatives). For each of these applications standard analytical columns (3.9 mm I.D.) and 1 ml/min flow-rates were employed with post-column stream splitting (1:100) before mass spectrometry. Various mobile phase compositions and solvent gradients were employed. The addition of 1–5% glycerol to the mobile phase was shown to have little effect on the chromatography. For all compounds studied (acylcarnitines, acyl-coenzyme A thioesters, and derivatized peptides) molecular weight information was obtained and sufficient sensitivity was achieved to allow unambiguous identification of trace components in complex mixtures.     

Analysis of gangliosides using fast atom bombardment mass spectrometry

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.     

Protein N-terminal analysis using fast atom bombardment mass spectrometry

An immunoassay is described that discriminates between monomers and oligomers of human leukocyte interferon. The assay in principle can be used to distinguish between monomers and oligomers of any substance.     

Detection of ribose-methylated nucleotides in Pyrodictium occultum tRNA by liquid chromatography—frit-fast atom bombardment mass spectrometry

Ribose-methylated dinucleotides of the type NmpN′ derived from digestion of tRNA with RNase T₂ were separated and characterized by directly combined liquid chromatography—mass spectrometry (LC—MS) with a continuous-flow frit-fast atom bombardment (frit-FAB) interface. Prediction of NmpN′ peaks was readily made by comparison of the LC profile with that of comparative nuclease P₁ digest. The identity of the candidate peaks including NmpN′ was further recognized by the mass spectra, in which NmpN′ showed intense molecular-related ions, in addition to sequence-specific fragment ions, to verify the chemical structures in both positive- and negative-ion modes. The method was applied to screening NmpN′ (and NmpN′mpN″) in tRNA from the extremely thermophilic archaeon Pyrodictium occultum.     

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.     

Post-translational processing of preprosomatostatin-II examined using fast atom bombardment mass spectrometry

The products and an intermediate of preprosomatostatin-II processing in the anglerfish islet were purified and subjected to structural analysis. The peptides isolated identify the site of signal cleavage (between Ser-24 and Gln-25). The prohormone is further processed at Arg-97 and, to a lesser extent, at the two adjacent basic amino acid residues Lys-61 and Arg-62. A 28-residue somatostatin is also generated which can be hydroxylated at Lys-23. A proteolytic processing site which would form the 14-residue somatostatin does not appear to be used to a significant degree. Fast atom bombardment mass spectrometry (FABMS) was used to demonstrate that the amino-terminal residues of peptides 25-60, and 25-90 are pyroglutamic acid, a modification which precludes Edman degradation of these peptides. Analysis of the peptides and tryptic peptide maps by FABMS allowed confirmation of the sites of prohormone conversion and indicated that terminal basic residues were removed during processing. Three amino acid residues were also found to differ from the amino acid sequence deduced from the cDNA and were localized to specific regions by FABMS analysis. Residues found to differ from the cDNA (cDNA in parentheses) were: Asp-77 (Thr), Val-78 (Phe), and Gly-90 (Glu). Mass assignments were confirmed by running a single cycle of Edman degradation prior to FABMS. The peptides noted above were also examined by Edman sequence analysis. The sequence of a cDNA clone to preprosomatostatin-II was re-examined in light of the observed differences at the protein level. This study emphasizes the utility of FABMS in prohormone processing studies and in identification of post-translational processing events.     

Derivatization of ketosteroids for fast atom bombardment mass spectrometry

Girard's reagents were used to derivatize ketosteroids and conjugates for analysis by positive ion fast atom bombardment mass spectrometry. Spectra contain an abundant ion corresponding to the cation (C+) of the newly formed ionic derivative (C+A-) and relatively little fragmentation. With derivatization, detection of ketosteroids at a concentration of 1 microgram microliter-1 in glycerol was straightforward. Such derivatization schemes may prove useful in the analysis of ketosteroids in complex biological mixtures.     

Identification of trichothecenes by thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography—mass spectrometry

Thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography—mass spectrometry are compared for the identification of six trichothecenes. Thermospray spectra of the trichothecenes exhibit only a very abundant ammonium adduct ion. Plasmaspray, which provides a more energetic ionization process than thermospray, produces some fragment ions in addition to an abundant ammonium adduct ion. The spectra obtained by dynamic fast-atom bombardment exhibit a protonated molecule, a glycerol adduct ion and numerous fragment ions formed by the losses of functional groups as neutrals in various combinations. Thermospray and plasmaspray are suitable only for monitoring of the trichothecenes, whereas dynamic fast-atom bombardment is suitable for monitoring and for structure characterization.     

Quantitative analysis of the kinetics of phospholipase A2 using fast atom bombardment mass spectrometry

Fast atom bombardment mass spectrometry that can directly analyze lysophospholipids was used to quantitatively determine the kinetics of phospholipase A2. This method is 1250 times more sensitive than the colorimetric assay.     

Application of fast atom bombardment mass spectrometry to chlorogenic acids

The technique of positive- and negative-ion fast atom bombardment mass spectrometry has been shown to be capable of producing molecular mass and useful fragmentation information for the structural elucidation of chlorogenic acids. The mass spectra of chlorogenic acid and the related compounds 3′-O-methylchlorogenic acid, neochlorogenic acid, 4,5-dicaffeoyl quinic acid and 1,5-dicaffeoyl quinic acid are compared with those obtained by electron impact mass spectrometry.     

C-terminal peptide identification by fast atom bombardment mass spectrometry.

A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.     

Positive ion fast atom bombardment mass spectrometry of some small oligosaccharides

Positive ion fast atom bombardment mass spectrometry appears to be a very useful method for the determination of molecular weight and composition of underivatized di- and trisaccharides. Information on the identity of the monosaccharide units can be obtained from the metastable ion and collisional activation spectra of selected ions. The type of linkage between the monosaccharides is reflected in some spectral characteristics, but the differences are relatively small and do not always allow an unambiguous identification. The position of a fructose unit in a trisaccharide molecule is shown by the collisional activation spectra of the [M + H]+ ion, as an anhydrofructose molecule is easily eliminated from the ions in which fructose is in a terminal position.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

Ribosyl-diphthamide: Confirmation of structure by fast atom bombardment mass spectrometry

Diphtheria toxin inactivates protein synthesis elongation factor 2 by attaching ADP-ribose to an unusual post-translational amino acid derivative, diphthamide, in the factor. Previously, we prepared ribosyl-diphthamide from the ADP-ribosyl-factor and proposed on the basis of NMR spectral analysis that it is 1-α-d-ribofuranosyl-2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine [N. J. Oppenheimer, and J. W. Bodley, (1981) J. Biol. Chem.256, 8579–8581 and op. cit.]. Now, using fast atom bomardment mass spectrometry, the intact cation of ribosyl-diphthamide has been observed in the gas phase. The theoretical mass of the structure proposed for ribosyl-diphthamide uniquely agrees with the observed mass of the inact cation of the compound to within 2 ppm. Collisional activation decomposition mass spectral analysis provided additional structural confirmation. Thus, although the compound has not been synthesized, all available evidence appears uniquely consistent with the structure of ribosyl-diphthamide previously proposed.     

Negative ion fast atom bombardment mass spectrometry for native gangliosides using a neutral matrix

A hexamethylphosphoric triamide proved to be a useful solvent for negative ion fast atom bombardment mass spectrometry (FABMS) of underivatized gangliosides, using a conventional glycerol matrix. Analysis of the gangliosides using a hexamethylphosphoric triamide was more convenient and more efficient not only for molecular weight determination but also for elucidating the structure of the carbohydrate sequence. We also noted the significance of the high polarity of the solvent as well as the electron pair donicity of the matrix system for negative ion FABMS of underivatized gangliosides.     

Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry

A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.     

Determination of plasma propofol levels using gas chromatography—mass spectrometry with selected-ion monitoring

Propofol (2,6-diisopropylphenol, I.C.I. 35 868) is a rapid-acting, intravenous anesthetic agent recently introduced for the induction and maintenance of general anesthesia. This paper describes a gas chromatographic—mass spectrometric procedure using selected-ion monitoring for the determination of plasma propofol levels. The drug and the internal standard (thymol) were extracted from plasma into diethyl ether—pentane, and derivatized to their trimethylsilyl derivatives before analysis. The reproducibility of the daily standard curves had coefficients of variation ranging from 2.7% to 10.2%. The precision of the assay yielded a coefficient of variation ranging from 4.5% to 5.6%, and the concentration means for the seeded control samples were found to be within −1.6% to +0.6% of the theoretical values for propofol. No interfering peaks have been observed in application of this procedure to either normal volunteer or patient samples. The minimum detectable level under the conditions described was 0.20 ng propofol/ml plasma. This assay and a high-performance liquid chromatographic assay with fluorescence detection were both used to measure plasma propofol concentrations in 89 human plasma samples, and the correlation between the two methods was excellent.