Coaxial continuous flow fast atom bombardment for higher-molecular-weight peptides: comparison with static fast atom bombardment and electrospray ionization.

A comparison of coaxial continuous flow fast atom bombardment (FAB) with static FAB and with electrospray ionization (ESI) for the analysis of 'high'-mass peptides (Mr = 3000-4000) is presented. Sensitivities of the peptides by coaxial continuous flow FAB is nearly an order of magnitude better than by static FAB. Single-scan spectra with good signal-to-noise can be obtained from as little as 200 fmol (by flow injection analysis). Detection limits by ESI mass spectrometry were found to be equivalent to 20 times higher than by coaxial continuous flow FAB on a per mole basis, but 4-20 times lower on a concentration basis, owing to the greater flow per unit time employed in the ESI mass spectrometric experiments.     

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.     

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.     

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.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

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.     

The analysis of multiple O-phosphoseryl-containing peptides by fast atom bombardment mass spectrometry

Summary Positive and negative ion FAB mass spectrometry were found to be useful for the structural analysis of phosphorylated peptides containing multiple O-phosphoseryl residues. The positive ion FAB mass spectra obtained for Ac-Ser(P)-Ser(P)-NHMe and Ac-Ser(P)-Ser(P)-Ser(P)-NHMe showed that -eliminative loss of H₃PO₄ from the Ser(P)-residue was a major event in the fragmentation of the two phosphopeptides and that successive losses of H₃PO₄ from the [M+H]⁺ ion occurred when the Ser(P)-cluster was located at the N-terminus. In contrast, the FAB mass spectrum of Ac-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe showed only a single loss of H₃PO₄ from the [M+H]⁺ ion, with further losses of H₃PO₄ from internal Ser(P)-residues only occurring when fragmentation of the parent phosphopeptide generated daughter fragments that contained (part of) an N-terminal Ser(P)-residue. Negative ion FAB mass spectrometry also proved useful for the structural analysis of the three Ser(P)-peptides and showed high-intensity [M-H]^- ions along with minor [M-H-80]^- fragment ions.Abbreviations Ac acetyl - Ala dehydroalanyl - FAB-MS fast atom bombardment mass spectrometry - LSIMS liquid secondary ion mass spectrometry - NHMe N-methylamide - Ser(P) O-phosphoseryl - Thr(P) O-phosphothreonyl     

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.     

Quantitative analysis of an N-oxide metabolite by fast atom bombardment tandem mass spectrometry

An assay for the N-oxide metabolite of a benzazepine drug by fast atom bombardment ionization with tandem mass spectrometric analysis on a triple quadrupole mass spectrometer has been developed and validated for urine and plasma samples. This methodology allows analysis of this metabolite directly in crude sample extracts, without the need for extensive chromatography or sample derivatization. Quantification was accomplished with the use of a stable isotope analog of the analyte as an internal standard, using the selected reaction monitoring mode of operation.     

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.     

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.     

Novel fast atom bombardment mass spectrometric procedures for glycoprotein analysis

We describe procedures, based upon fast atom bombardment mass spectrometry, for characterising the carbohydrate chains in glycoproteins and for determining sites of glycosylation. Strategies for rapidly screening glycoproteins to ascertain the types of sugar chains present and the degree of heterogeneity are presented. Protocols are given for sequencing O- and N-linked glycans. These strategies exploit simple purification steps and afford data at high sensitivity.     

Synthesis of a cyclic fibrin-like peptide and its analysis by fast atom bombardment mass spectrometry

For immunochemical purposes, a cyclic 12-peptide was synthesized to model the gamma-gamma-chain cross-link site in human fibrin. The model was based upon the structure proposed by Chen & Doolittle (Biochemistry (1971) 10, 4486-4491) which is characterized by two reciprocating epsilon-(gamma-Glu)Lys bonds between adjacent fibrin gamma-chains oriented in an antiparallel manner. To achieve the antiparallel orientation of the peptide backbone, Pro and Gly were inserted at positions 6 and 7 of the linear 12-peptide: acetyl-Gly-Glu-Gln-His-His-Pro-Gly-Gly-Gly-Ala-Lys-Gly-amide. The insertions were made to facilitate a reverse turn of the peptide during the last synthetic step, which was formation of the epsilon-(gamma-Glu)Lys bond between Glu at position 2 and Lys at position 11 with diphenylphosphorylazide. The resulting cyclic peptide represented half of the symmetrical cross-linked region in clotted fibrin. Following purification by HPLC, both linear and cyclic 12-peptides were analyzed by fast atom bombardment mass spectrometry. Abundant molecular protonated ions were observed for both peptides. In addition, the amino acid sequence of the linear peptide and the location of the epsilon-(gamma-Glu)Lys bond in the cyclized peptide could be verified.     

Structural and mixture analysis of glycerophosphoric acid derivatives by fast atom bombardment tandem mass spectrometry

It is shown that by a combination of positive and negative FAB mass spectrometry with collision activation using a tandem mass spectrometer diacyl glycerophosphoric acid ester mixtures can be analysed. The following results will be obtained: Molecular masses of the single components, nature of the residue bound to the phosphoric acid (choline, serine etc.), fatty acids present in the single components and (if different) their location at C-1/C-2 as well as a quantitative analysis both of the fatty acids in the mixture and of the various species making up the latter.     

Comparison of 252californium plasma desorption and fast atom bombardment mass spectrometry for analysis of small peptides

The data obtained with 252Cf plasma desorption (PD) and fast atom bombardment mass spectrometry of eight tri-, tetra- and pentapeptides were compared. Good spectra were obtained with 1-10 nmol of peptide. In both techniques molecular weight information was obtained. The PD mass spectra are often dominated by the cationized molecular ions in contrast to the fast atom bombardment (FAB) mass spectra, where cationization is rarely observed. Amino acid content is reflected in the immonium ions equally well in both techniques. The fragmentation patterns observed with the two techniques are almost identical. However, practical sequencing of peptides based on either FAB or PD mass spectrometry of underivatized peptides alone is difficult. This is due to the unpredictable and sometimes absent cleavage yield at certain peptide bonds. Another difficulty is the many simultaneous fragmentation pathways. However, for many peptides enough information is present to allow sequence determination for at least a major part of the molecule.     

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.     

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.     

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.