Peptide studies using a fast atom bombardment high field mass spectrometer and data system. 3--Negative ionization: mass calibration, data acquisition and structural characterization

Under negative ionization conditions, nominal mass calibration of the fast bombardment high field mass spectrometer and data system was accomplished using cesium iodide/glycerol as a reference. Mass calibration at --8 kV accelerating potential extends from m/z 387 to m/z 2170 using xenon fast atoms. Negative xenon FAB mass spectra for human angiotensin I and human gastrin I complement their positive fast atom bombardment spectra. Negative xenon fast atom bombardment spectra of underivatized peptides exhibit molecular proton-abstracted ion envelopes and structurally significant fragment ions. Peptide mixture analysis under negative xenon fast atom bombardment reveals peptide molecular ion envelopes of higher relative intensities than under positive xenon fast atom bombardment.     

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.     

Determination of the amino acid sequence of an intramolecular disulfide linkage-containing sperm-activating peptide by tandem mass spectrometry.

A sperm-activating peptide (SAP) was isolated from the egg jelly of the sea urchin Stomopneustes variolaris. The presence of an intramolecular disulfide linkage in the peptide was demonstrated by fast atom bombardment (FAB) mass spectrometry with the intact and reduced peptides. The amino acid sequence of the reduced peptide was determined to be Lys-Phe-Cys-Pro-Glu-Gly-Lys-Cys-Val by tandem mass spectrometry from the spectrum produced by a collision-induced decomposition method. Furthermore, it was also demonstrated that SAPs obtained from sea urchins Arbacia punctulata and Glyptocidaris crenularis are cyclic peptides containing one cystine residue by FAB mass spectrometry.     

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.     

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.     

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.     

Primary structure of the hypertrehalosaemic factor II from the corpus cardiacum of the Indian stick insect, Carausius morosus, determined by fast atom bombardment mass spectrometry

The primary structure of hypertrehalosaemic factor II, isolated from the corpus cardiacum of the Indian Stick Insect Carausius morosus, has been assigned as Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 from its fast atom bombardment (FAB) mass spectrum and metastable scans of its FAB spectrum. The structure assigned shows close homology to other insect neuropeptides. A synthetic sample of this peptide gave the same FAB spectra, reversed-phase high-performance liquid chromatographic behavior, and biological behavior as the natural material. Mass spectrometric fragmentation of the synthetic peptide was examined by B/E linked scan and MIKES techniques in a two-sector mass spectrometer and by the MS/MS technique in a four-sector (tandem) spectrometer.     

Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fast atom bombardment mass spectrometric characterization of peptides

Structural characterization of peptides in the range of 500–5000 Da, using fast atom bombardment (FAB) and Cs⁺ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include syntheitc peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article.     

The electron impact, chemical ionization and fast atom bombardment positive ion mass spectra of 1,2-bis(sulfonyl)methylhydrazines.

A series of bis(sulfonyl)-1-methylhydrazines were analyzed by positive ion electron impact (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry. Since these compounds showed activity against the L1210 leukemia, an understanding of their mass spectral behavior is important should the structural characterization of metabolites be required. FAB proved to be the most useful technique, generally providing abundant protonated molecule ion peaks, in contrast to the weak peaks observed with CI (ammonia or isobutane) and the total absence of molecular ion peaks in the EI mass spectra. In addition, utilizing FAB eliminated the problem of thermal decomposition, which was very difficult to control under EI and CI experimental conditions. Fragments observed in FAB and CI mass spectra were consistent with protonation at the methyl-bearing nitrogen. One can locate the R1 and R2 moieties relative to the methyl-bearing nitrogen in FAB and CI by assigning that nitrogen as the site of protonation, with subsequent elimination of R2SO2H.     

Verification by mass spectrometry of the primary structure of human interleukin-2

Proteolytic digests of interleukin-2 from a human leukemic T-cell line produced by Escherichia coli carrying a recombinant DNA were analyzed by fast atom bombardment mass spectrometry. The mass values of intense signals observed in the mass spectrum were consistent with peptides predicted from the nucleotide sequence of cDNA for human interleukin-2, an indication that the protein with the predicted amino acid sequence was produced by E. coli. BrCN and proteolytic digests of interleukin-2 obtained from cultured cells were also examined by fast atom bombardment mass spectrometry. The observed mass values were identical with those from interleukin-2 from E. coli except for that of the NH2-terminal sequence, in which the Thr residue at position 3 was bound to a sugar moiety. The mass spectra of the digests of the two interleukin-2 preparations and synthetic peptides with sequences from 117 to 128 and 121 to 128 predicted from the nucleotide sequence of cDNA for a human interleukin-2 indicated that Cys residues at positions 58 and 105 are linked by a disulfide bond and that the Cys residue at position 125 is free.     

Fast ATOM Bombardment Mass Spectra of Nucleosides. Comparison with Electron Impact and Chemical Ionization Mass Spectra

Abstract

The fast atom bombardment (FAB) mass spectra of the eight major nucleosides found in RNA and DNA, pseudouridine and 2′,3′-O-isopropylidene adenosine are described and compared to El, CI, and desorption chemical ionization (DCI) spectra reported in the literature or obtained in this laboratory. Bcty, cocltl nun FAB spectra are reported. The FAB spectra are simple and provide unambiguous molecular weight information along with structurally significant fragment ions. Mechanisms of ion formation are thought to closely parallel those suggested earlier to be operating in the CI mode. Advantages and disadvantages of FAB relative to the standard ionization modes are discussed.     

Synthesis of cystine peptides 21-25/70-73 and 35-39/56-59 of the beta-subunit of human choriogonadotropin.

Syntheses of two asymmetrical cystine peptides with the amino acid residues 21-25/70-73 and 35-39/56-59, based on the linear amino acid sequence and the disulfide bond assignment in the beta-subunit of human choriogonadotropin (hCG-beta), are described. S-trityl and S-acetamidomethyl peptide fragments of each cystine peptide were prepared in solution phase and were subjected to oxidation with I2/MeOH to form the disulfide bridge. The cystine peptides were characterized by their amino acid analyses and fast atom bombardment mass spectrometry. Immunological characterization by several homologous radioimmunoassay systems showed that peptide 21-25/70-73 had significant hCG, hCG-beta, and hLH activities while peptide 35-39/56-59 failed to reveal any immunoreactivity.     

Fast atom bombardment tandem mass spectrometric analysis of N-carbamoylamino acids.

Using fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), we examined 12 synthetic N-carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives. In FAB mass spectrometry and FAB MS/MS, spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. The daughter scan spectrum of the parent ion indicated that it was useful for structural elucidation and differentiation of structural isomers of CAA. We have also identified each CAA separately in a mixture using a neutral loss scan for characteristic ions. In addition, we demonstrated that CAA in urine samples from patients with ornithine carbamoyl transferase deficiency gave collision-induced dissociation (CID) spectra which correspond well with CID spectra obtained using synthetically prepared CAA.     

Determination of interchain crosslinkages in insulin B-chain dimers by fast atom bombardment mass spectrometry

New methodology for identifying and locating crosslinkages in peptides is described. Pepsin is used to cleave insulin and B-chain dimers of insulin into fragments under conditions which retain the original peptide crosslinkages. After partial separation by HPLC, the peptides are analyzed by fast atom bombardment mass spectrometry (FABMS) to determine their molecular weights. The molecular weights of peptide fragments expected from the pepsin digests of human insulin and related model compounds are calculated from the amino acid sequence of the intact peptide. Digestion products are identified by matching their molecular weights, as determined by FABMS, with calculated molecular weights. Locations of interchain crosslinkages are deduced after the peptide fragments have been assigned to specific segments of the parent peptide. The validity of the method has been established by correctly identifying structurally important products in the pepsin digests of model compounds such as human, bovine, and porcine insulins. Procedures developed with the model compounds were used to identify crosslinkages in peptides of unknown structure isolated from an insulin A-chain/B-chain combination reaction mixture. Evidence is presented for formation of three different types of crosslinkages, disulfide, lanthionine, and sulfoxide.     

Structural analysis of biologically active peptides and recombinant proteins and their modified counterparts by mass spectrometry

The structural characterization of the Escherichia coli-expressed human interferon α-2b (rh-IFN α-2b) was carried out by employing the fast atom bombardment (FAB) and plasma desorption (PD) mapping methods. The mass spectral data of the rh-IFN α-2b and the trypsin-generated peptide mixture allowed rapid and facile confirmation of the cDNA-derived sequence and determination of the existing disulfide pattern in the protein molecule. The same PD/FAB mapping approach was successfully employed in the structural determination of the iodination reaction product of rh-IFN α-2b and the potent vasoconstrictor peptide endothelin.     

Physalaemin-like immunoreactive peptides from rabbit stomach

Two physalaemin (PHY)-like immunoreactive peptides, designated PHLIPs, have been purified from extracts of rabbit stomach tissue. Fast atom bombardment/mass spectrometry (FAB/MS) indicated that the m/z values for the PHLIP protonated molecular ions were 867.419 and 796.4. FAB/tandem MS spectra, coupled with a knowledge of the amino acid composition and the aid of a computerized fragment-matching program, indicated the amino acid sequences to be: (formula; see text) The sequences of PHLIPs-7 and -8 were confirmed with synthetic peptides. The PHY-antiserum cross-reactivity of the PHLIPs reflects homology at amino acid residues 1, 3, 4 and 5 for the mammalian and amphibian residues.     

Glutamyl-Taurine Is the Predominant Synaptic Taurine Peptide

Several taurine-containing peptides have been identified from trichloracetic acid extracts of synaptosomes and their subcellular vesicles prepared from calf brain. These peptides contain aspartic and glutamic acids, serine and taurine, and are often present in an N-acetylated form. The peptides were isolated as single spots by TLC. Glutamyl-taurine was found to be the predominant structure when analyzed by fast atom bombardment (FAB) mass spectrometry.     

Structural characterization of lichenysin A components by fast atom bombardment tandem mass spectrometry.

The structural characterization of the cyclic lipoheptapeptide surfactant lichenysin A components, produced by Bacillus licheniformis strains via the non-ribosomal pathway on a corresponding peptide synthetase, was carried out using a tandem mass spectrometry (MS/MS) under fast atom bombardment (FAB) conditions. Based on the analysis of the collision-induced fragment-ion spectrum of the single charged molecular ions of both native and partially hydrolyzed forms of lipopeptide, a new general structure of lichenysin A components was elucidated. It varies from previously proposed structure by having in the peptide portion of lipopeptide the L-Gln-1 and L-Asp-5 residues instead of L-Glu-1 and L-Asn-5. The verified chemical structure of lichenysin A was found to be reflected in the structural organization of the corresponding lichenysin A synthetase, LchA, described recently.