Substance P self-aggregation revised: a chromatographic and mass spectrometry analysis

Size exclusion liquid chromatography and electrospray ionization mass spectrometry have been used for the first time to analyze the possible effect of the environmental conditions on substance P (Arg-Pro-Lys-Pro-Gln-Gln- Phe-Phe-Gly-Leu-Met-NH2) self-association. The effects of different parameters such as solvent ionic strength, pH and polarity have been evaluated by monitoring the changes observed in aggregation forms registered by size exclusion chromatography. Mass spectrometry has been employed with the aim of identifying the size of the aggregation clusters formed in gaseous state. These two techniques can be considered complementary since they allow describing the same phenomena by different points of view permitting to identify the possible role of environmental parameters in controlling the distribution of different sized aggregates. Thus, new details can be added to the previous results reported by other methods. The results obtained indicate that substance P was associated under all conditions tested and that very limited variations of the conditions under which substance P was solubilized were accompanied by perceptible changes in the aggregation figures. Together with existing data, the data obtained have been interpreted in terms of a bi- or multi-stage model according to which an initial stepwise aggregation taking place through successive additions of single molecules is followed by the coalescence of the already-formed aggregates into larger complexes. Such a model appears to be able to explain the large variations in SP aggregation figures, and the high sensitivity of these figures to limited modifications of environmental parameters indicated by the data obtained.