Site-specific sodiation of peptides studied by pulsed nanoelectrospray ionization

Abstract

Metal ions play significant roles in biological systems. Although sodium ions play a key role in biological systems, site-specific interactions between sodium ions and protonated peptides and proteins have not been thoroughly explored. In this work, the time-dependent sodiation of protonated peptides was investigated by pulsed nanoelectrospray ionization. Mass spectra for aqueous mixtures of 5×10−6 M angiotensin II (A), bradykinin (B), and gramicidin S (G) were measured as a function of the high voltage (4000 V) pulse width (PW) with and without the application of a bias voltage (BV) to the solution. With BV = 0 V, only [B + 2H − H + Na]2+ was detected with an increase in PW from 220 μs (threshold for the signal appearance) to 1 ms. In contrast, [A + 2H − nH + nNa]2+ (n = 1–3) started to be detected with PW = 500 μs and up to n = 4 with PW = 1 ms. By the application of BV = 900 V, [B + 2H − nH + nNa]2+ with n = 0 and 1 started to be detected with PW = 40 μs. With further increase in PW up to 1 ms, [B + 2H − nH + nNa]2+ with n = 0–3 and [A + 2H − nH + nNa]2+ (n = 1–5) were detected. The observation of [B + 2H − 3H + 3Na]2+ and [A + 2H − 5H + 5Na]2+ with BV = 900 V suggests that carboxyl (−COOH), imidazole > N–H, hydroxyl (−OH) as well as amide > N–H bonds are deprotonated to form respective sodiated forms of carboxylate –COO−⋅⋅⋅⋅Na+, imidazole > N−⋅⋅⋅⋅Na+, hydroxyl –O−⋅⋅⋅⋅Na+ and amide –NC–O−⋅⋅⋅⋅Na+ bonds. Ions of G were totally suppressed by the presence of A and B in aqueous solution.