Mass spectrometry of proteins and peptides

Abstract

In the last few years, mass spectrometry (MS) has emerged as a major tool for the identification and characterization of peptides and proteins. It is now possible to measure proteins of masses > 100 kDa to an accuracy of a few Da, and to measure the masses of peptides with an accuracy of a few mDa. As a result, the information yielded by an MS measurement is highly specific. In addition, MS has extremely high sensitivity, often in the femtomole (10?15 mole) range, so it is a method that is suitable for the analysis of trace amounts of sample. Mass spectrometry relies on the properties of charged particles moving under the influence of electric and magnetic fields, so the species studied must be ions (charge ze) rather than molecules. Most MS experiments are performed on positive ions, which can be formed either by the removal of one or more electrons from each molecule, or by the addition of one or more cations, usually protons. Consequently, the first step in any mass measurement is to transfer the molecule into the gas phase and to ionize it; what is actually measured then is the ratio of molecular mass to charge (m/z). Most biological samples are mixtures of a number of compounds, so their m/z spectra can be very complex. Moreover, even pure compounds yield a number of peaks in their m/z spectra, corresponding to various values of charge z, and to the distribution of isotopes in the compound. The main isotope effect in organic compounds is caused by the ~1.1% 13C in natural carbon. For example, Fig. 27.1 shows the m/z spectra of two singly charged peptide ions. In Fig. 27.1A the most abundant peak in the spectrum is the "monoisotopic" peak at 1053.587 Da (see definitions in Table 27.1), corresponding to the ions containing only the most abundant isotopes. For larger molecules, such as the peptide shown in Fig. 27.1B, the distribution is shifted upwards, and the monoisotopic peak is no longer the largest one. As the molecular mass increases still further, the probability that all atoms in the molecule consist of the most abundant isotopes decreases, so the monoisotopic peak continues to decrease until finally (at >15 kDa) it is usually too small to observe. © 2008 Humana Press.