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The development of mass spectrometric methodologies for determining the amino acid sequences of peptides and the primary structure of proteins began in the late 1950s with the conversion of small peptides to analogous polyamino alcohols that were sufficiently volatile to be separated by gas chromatography and ionized by electron impact. Sequencing was greatly simplified by the ability to ionize intact peptides and even entire proteins directly afforded by the invention of FAB, MALDI, and ESI in the 1980s. The advent of DNA sequencing provided further synergistic avenues to the efficient determination of the primary structure of even very large proteins.

Publisher Summary This chapter gives an overview and strategy for peptides and proteins. There are many problems in peptide and protein structure that can be simply solved by determining molecular weights to better than one mass unit. One of the first applications of fast atom bombardment- mass spectrometer (FAB-MS) to protein structure was the verification and, if necessary, correction of the DNA sequence of a gene coding for the corresponding protein. It merely requires translating the open reading frame of the gene sequence into the corresponding protein sequence and calculating the molecular weights of the tryptic peptides expected from the locations of lysine and arginine. Matching these values with the actual molecular weights, determined by FAB-MS, of the components of the tryptic digest of the protein reveals whether or not the DNA is correct. Those values that do not match indicate either an error in the DNA sequence or a posttranslational modification of the gene product. Sequence errors are usually misidentifications of a base that leads to a codon representing a different amino acid, or a deletion or insertion of a base causing a frame shift.

Publisher Summary This chapter discusses utility of exact mass measurements. High-resolution mass spectrometry and exact mass measurement have been synonymous with the determination of elemental composition of an organic compound or a fragment ion ever since Beynon, in the early 1960s, demonstrated the practical utility of these techniques. By precisely measuring (to about 1–10 ppm) the relative ratio of the accelerating voltages required to focus, at fixed magnetic field, an ion of unknown mass and a reference ion onto the collector slit of a double-focusing mass spectrometer, it is possible to measure the m/z value of the unknown ion with an accuracy that permits the calculation of its elemental composition. Such measurements serve a variety of purposes: the elucidation of fragmentation pathways of compounds of known structures; the determination of the elemental composition of synthetic compounds in lieu of combustion analysis; the determination of the structure of natural products; etc. Both the information content of an exact mass measurement and the experimental difficulties in obtaining the data, change dramatically as the mass of the molecule or the fragment increases.

Publisher Summary This chapter discusses sequencing of peptides by tandem mass spectrometry and high-energy collision-induced dissociation. To ionize large polar molecules, such as peptides of a size encountered in the study of various aspects of protein structure, methods are used that impart little excess energy on the molecule. Furthermore, most of these "soft" ionization techniques produce the charged particle by addition or removal of a proton to form the very stable, even-electron ions (M + H) + or (M - H) - , that have little tendency to fragment. The first demonstration of the generation of large protonated peptides involved fast atom bombardment (FAB), but the data and methodology discussed in this chapter utilize either Xe ° or Cs + as primary particles. Peptides derived from proteins are linear molecules (at least after reduction of any disulfide bridges originally present) that consist of repeating units having an identical backbone, –NH–CH(R)–CO–, but differing in the nature of the side chain, R. There are only 20 different amino acids that occur in proteins (and are defined by the genetic code), in addition to the occasional occurrence of others formed by posttranslational processes.

Krypton and xenon have been discovered in the Martian atmosphere with the mass spectrometer on the second Viking lander. Krypton is more abundant than xenon. The relative abundances of the krypton isotopes appear normal, but the ratio of xenon-129 to xenon-132 is enhanced on Mars relative to the terrestrial value for this ratio. Some possible implications of these findings are discussed.

Publisher Summary This chapter illustrates strategies and methods for identification of posttranslationally modified amino acids in proteins by mass spectrometry. Mass spectrometry (MS) is particularly well suited for the investigation of compounds whose structure is unknown and for which reference compounds do not exist. The particular MS method employed is dictated by the level of information desired as well as the particular instrumentation available. Mass spectrometric protein sequencing strategies is divided into two broad categories—namely, (1) those which employ conventional ionization methods, such as electron impact (EI) or chemical ionization (CI), and require that the amino acid or polypeptide be chemically derivatized and (2) those which are capable of directly analyzing underivatized large or polar molecules–the soft ionization methods. The soft ionization methods eliminate the need for prior chemical derivatization which may destroy sensitive functional groups, requires more material and is often tedious. Fast atom bombardment mass spectrometry allows tackling much larger molecules such as peptides up to 10,000 daltons.