Analysis of Neuropeptides by Size-Exclusion HPLC Linked to Electrospray Ionization Mass Spectrometry

Atmospheric pressure ionization (API) interfaces have become powerful and popular tools for sample ionization (so-called soft ionization) and are primarily used in the analysis of polar and thermolabile compounds (e.g., peptides, proteins). Detailed descriptions of the most important features of this technique have been published elsewhere (1 ,2 ). The API technique is based on the ionization of the sample in solution passing a thin capillary, and applying high potential (2–5 kV) at the capillary tip. This process causes formation of the small, charged droplets, containing both solvent and the sample. After evaporation of the solvent (desolvation), the dry ions in the gas phase are directed to the ion source, accelerated, and analyzed. Every mass spectrometer analyzes mass-to-charge ratio (m/z) rather than the molecular mass of the compound. In other words, addition of one proton to the molecule of a nominal molecular weight of 2000 Dalton, will result in detection of a singly-charged ion at m/z of 2001/1 = 2001. Attraction of, e.g., 4 protons to this molecule will cause detection of the quadruply-charged ion at m/z of 2004/4 = 501. This observation leads to the conclusion that, using API interfaces, it would be possible to detect molecules like proteins or longer polypeptides having molecular masses that are far beyond the scanning range of instruments, i.e., >3–5 kDa. The maximal number of attracted charges depends on the amount of polar amino acids within the analyzed molecule. In fact, molecules are represented by several multiply charged species, constituting mass spectrum (Fig. 1A ), which, in turn, needs to be transformed to the molecular mass (so-called deconvolution process) as shown in Fig. 1B .