Practical considerations in analysing neuropeptides, calcitonin gene-related peptide and vasoactive intestinal peptide, by nano-electrospray ionisation and quadrupole time-of-flight mass spectrometry: monitoring multiple protonations.

We investigated the pre-electrospray ionisation (pre-ESI) factors; analyte concentration (1-2500  ng/mL), concentration of formic acid (FA) in the mobile phase (0.01, 0.1 and 1%), concentration of the organic modifier (acetonitrile 50-90%) and flow rate (<10  μL/min) on the number of multiple protonations and ESI response for two neuropeptides (of ~3.3  kDa molecular mass); calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP). A pH of 3.23 (0.1% FA), nano-flow rate range of 350-750  nL/min and acetonitrile concentration of 50% were optimum for both neuropeptides where the highest intensities were observed. An inverse relationship between decreasing flow rate and ESI response for both peptides was also observed. The quadruply charged ([M+4H](4+)) ion was dominant for CGRP at all analyte concentrations, and also for VIP, but only at the higher analyte concentrations (250-2500  ng/mL); none of the [M+4H](4+), [M+5H](5+) or [M+6H](6+) ions were dominant at the lower concentrations. Linear correlations were obtained for the protonated states and ESI response at analyte concentrations (1-750  ng/mL). Acetonitrile concentration was critical; severe ion suppression was observed for VIP when the concentration of acetonitrile was ≥60%. Ion suppression was also observed for both peptides in an equimolar mixture, with the extent of ion suppression more severe for VIP. Our study concludes that it is important to monitor several protonated species when a single protonated state does not dominate, especially during label-free peptide quantitations.