Exploring Deprotonation Reactions on Peptides and Proteins at Atmospheric Pressure by Electro-Sonic Spray Ionization-Mass Spectrometry (ESSI-MS)

Electrospray ionization mass spectrometry (ESI-MS) rapidly became established as the method of choice for the production of large biomolecular ions in the gas phase. For peptides and proteins, ESI leads to the formation of multiply charged ions, in both the positive and negative ion mode. The charge-state distribution is directly related to the conformation of the macromolecular ions and to proton transfer reactions in the gas phase. Deprotonation reactions of multiply charged peptide and protein ions were studied by introducing volatile reference bases at atmospheric pressure between an electro-sonic spray ionization (ESSI) source and the inlet of a mass spectrometer. This new set-up offers the unique possibility to measure the apparent gas-phase basicity GBapp of multiply charged ions by a bracketing approach. The proof of principle was made using bradykinin derivatives, substance P and insulin chain B. We obtained values in excellent agreement with known GBapp values obtained at low pressure. These experiments were extended to seven model proteins showing that the thermodynamical properties of protein ions are directly correlated to the amino-acid sequence and the conformation in the gas phase. We also demonstrated that salt bridges between ionized basic and acidic sites still exist in the gas phase, confirming that electro-sonic spray is a very soft ionization technique.