Mass spectrometry (MS) has become a vital enabling technology in the life sciences. This chapter summarizes the fundamental aspects of MS, with reference to topics such as isotopic abundance and accurate mass and resolution. A broad and comprehensive overview of the instrumentation, techniques, and methods required for the analysis of biomolecules is presented. Emphasis is placed on describing the soft ionization methods and separation techniques employed in current state-of-the-art mass Spectrometers.As defined in a publication from the International Union of Pure and Applied Chemistry (IUPAC), MS (or mass spectroscopy) is "the study of systems by the formation of gaseous ions, with or without fragmentation, which are then characterized by their mass-to-charge ratios and relative abundances" (Todd, 1991). Since the publication of the last volume in Methods in Enzymology reviewing MS of biomolecules (McCloskey, 1990), there has been a revolution in the field. Two promising novel soft ionization methods emerging at that time were not generally available, partly because both were largely incompatible with the typical commercial sector mass Spectrometers that were in widespread use. Although the particle bombardment/desorption techniques of plasma desorption MS (PDMS), fast atom bombardment (FAB), and liquid secondary ion MS (LSIMS), invented a decade earlier, had been making valuable contributions to the analysis of peptides, oligosaccharides, and other polar and involatile compounds, they were largely limited to the picomole range and thus lacked the sensitivity needed to tackle the most challenging problems. During that period when analysis of intact biological molecules such as small proteins first became possible, much research was focused on attempts to ionize ever larger molecules, many of which were standards purchased from commercial suppliers. With hindsight, simply measuring the molecular weight of a large molecule is often of limited utility, whereas digesting it chemically or enzymatically to smaller moieties and measuring the masses of even a subset of these can be very informative. Today, thanks to the maturation of soft ionization methods and new developments in mass analyzers optimized for these new ionization methods, MS steps in developing reliable quantitative methods in biological MS, this still presents substantial difficulties. Thus, looking ahead the immediate challenges are to analyze larger numbers of samples within shorter periods of time, achieve even higher sensitivity of detection, and improve the ability to quantitate the molecules under investigation. This will require enhanced data processing and interpretation, with validated scoring methods that will allow automation to the degree that reliable results will be generated by computerized methods with little or no human input.

Liquid chromatography/electrospray ionization mass spectrometry was used to investigate the peptide composition of the venom of Conus pennaceus, a molluscivorous cone shell from the Red Sea. Based on observed M(r)s, this venom contained all known conotoxins previously isolated and identified from this species. Interestingly, the doubly protonated species of only two of these conotoxins, alpha-PnIA and alpha-PnIB, showed additional related ions at +40 m/z (+80 Da), indicating the presence of either sulfation or phosphorylation in both components. High-performance liquid chromatographic (HPLC) fractions containing these two conotoxins were examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in both positive and negative ion modes, as well as by MALDI high-energy collision-induced dissociation. These experiments established the presence of a single sulfated tyrosine residue within both alpha-PnIA and alpha-PnIB. Hence their post-translationally modified sequences are GCCSLPPCAANNPDY(S)C- NH2 (alpha-PnIA) and GCCSLPPCALSNPDY(S)C-NH2 (alpha-PnIB). This assignment was supported by comparison of their mass spectral behavior with that of known sulfated and phosphorylated peptides. This data clarified further the distinguishing features of the ionization and fragmentation of such modified peptides. Selective disulfide folding of synthetic alpha-PnIB demonstrated that both sulfated and non-sulfated toxins co-elute on reversed-phase HPLC and that alpha-PnIB possesses the same disulfide connectivity as other 'classical' alpha-conotoxins reported previously

Each amino acid in a peptide has a characteristic immonium ion (H2N+ = CHR), the presence of which in a mass spectrum can indicate the presence of that amino acid. High-energy collision-induced decomposition studies on small peptide ions formed by fast atom bombardment showed the relative intensities of these immonium ions to be dependent on the relative positions of the amino acids in the peptide chain: C-terminal, N-terminal or in-chain. Evidence in favour of competition in the formation of immonium ions is presented.

ChemInformVolume 19, Issue 24 Physical Organic Chemistry ChemInform Abstract: Tautomerism in Aromatic Hydroxy N-Heterocyclics in the Gas Phase by Metastable Ion Mass Spectrometry M. A. BALDWIN, M. A. BALDWIN Sch. Pharm., Univ., London WC1N 1AX, UKSearch for more papers by this authorG. J. LANGLEY, G. J. LANGLEY Sch. Pharm., Univ., London WC1N 1AX, UKSearch for more papers by this author M. A. BALDWIN, M. A. BALDWIN Sch. Pharm., Univ., London WC1N 1AX, UKSearch for more papers by this authorG. J. LANGLEY, G. J. LANGLEY Sch. Pharm., Univ., London WC1N 1AX, UKSearch for more papers by this author First published: June 14, 1988 https://doi.org/10.1002/chin.198824056Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume19, Issue24June 14, 1988 RelatedInformation

AbstractAb initio molecular orbital calculations have been used to determine the degree of charge localization associated with the formation of radical cations on electron impact of formamide, thioformamide and their N‐methyl and N, N‐dimethyl analogues. The charge favours the nitrogen of formamides and the sulphur of thioformamides, but the degree of localization is calculated to be lower than in the radical cations of urea and thiourea.

A chiral effect has been observed for formation of protonated dimers of dialkyl tartrates under fast atom bombardment, analogous to the chemical ionization behaviour reported previously. Homochiral dimers show greater stability than the heterochiral dimers, as is evidenced by their enhanced formation and lesser tendency to undergo metastable breakdown. The difference in stability is related to the size of the alkyl group.

AbstractExtensive networks of metastable ions link the major peaks in the electron impact mass spectra of two crown ethers containing 2,6‐pyrido units. High‐resolution mass measurements and the metastable peaks allow the elucidation of the fragmentation pathways. The spectra are influenced more by the presence of aromatic substituents than by the 2,6‐pyrido units.

AbstractCritical energy measurements on 13C‐labelled quinolines show that the energy requirement for elimination of H(C‐2)N from metastable ions is lower than that for H(C‐3)N elimination, the critical energies being 4.03 ± 0.05 eV and 4.17 ± 0.05 eV, respectively. It is also shown that involvement of C‐2 and C‐3 in unimolecular HCN elimination from ions in the second field‐free region is greater, by factors of approximately 3 and 2 respectively, than would be anticipated for elimination following complete carbon randomization.

Abstract[2‐13C]Quinoline and [3‐13C]quinoline were synthesised by a five‐step process starting from isatin. Acetylation of isatin with acetyl chloride labelled in either the 1‐ or 2‐position, was followed by the pfitzinger reaction to give 2‐hydroxy‐4‐quinoline carboxylic acid labelled in the 2‐or 3‐position. Decarboxylation by pyrolysis was followed by chlorination and reduction to the labelled quinolines. The overall yield was approximately 20%

Abstract13C labelling has been used to study isoquinoline molecular ions undergoing breakdown by HCN elimination in a mass spectrometer. For otherwise stable ions caused to fragment by collisional activation, there is no skeletal rearrangement prior to HCN loss. Of the ions formed by 70 eV electron impact, 69% of those which fragment in the ion source by HCN loss retain their structural integrity, as do 44% of the metastable ions. Of the ions that eliminate HCN without prior arrangement, approximately two‐thirds eliminate C‐1 and one‐third eliminate C‐3. Critical energies are reported for the elimination of HCN from pyridine and isoquinoline molecular ions.

AbstractThe structures of the [MOH]+ ions of m‐ and pethylnitrobenzene have been compared by measurements of metastable ion spectra, collisional activation spectra, kinetic energy releases and critical energies for the formation of these ions and their subsequent decomposition. Normalized rates of fragmentation of metastable molecular ions and metastable [MOH]+ ions have been compared for ion lifetimes up to 30 μs. The energy measurements fail to distinguish between the structures of the [MOH]+ ions, but the normalized fragmentation rates and the collisional activation spectra show their structures to be different.

AbstractThe loss of a hydroxyl radical from the molecular ions of o‐, m‐ and p‐ethylnitrobenzene has been studied by metastable ion and collisional activation techniques using electron impact and field ionization. It is shown that for the ortho isomer the mechanism of this reaction is unique, and is totally different from that of the meta and para isomers. The critical energies are also reported, and deuterium labelling is employed to access the role of the α‐hydrogens in hydroxyl loss.

A series of bruceolides isolated from Brucea javanica has been studied by electron impact, chemical ionization and field desorption mass spectrometry, and by metastable ion techniques, including mass analysed ion kinetic energy spectrometry. The spectral data obtained for one of these compounds, bruceine A, are discussed in relation to the molecular structure and are taken as an example for the bruceolide series. The bruceolides occur in plants as mixtures of closely related compounds and some of them have potent anti-cancer activity. It is demonstrated that mass spectrometry provides a valuable tool for the identification and characterization of these compounds.

Measurement of the appearance energy of the [C6H4]+⋅ ion from benzonitrile molecular ions fragmenting in the second field free region of a reverse geometry double focusing mass spectrometer, gives a value free of any significant kinetic shift. The heat of formation of the [C6H4]+⋅ ion calculated using this appearance energy is 1348 kJ mol−1, which is consistent with the ion having the benzyne structure.