Abstract
Deuterium exchange was monitored by electrospray ionization mass spectrometry (ESI‐MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several α‐helical peptides and β‐sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, α‐helical, or β‐sheet structure. For 3 isomeric 31‐residue α‐helical peptides, the number of slowly exchanging hydrogens as measured by ESI‐MS in 50% CF~3~CD~2~OD (pD 9.5) provided estimates of their α‐helicities (26%, 40%, 94%) that agreed well with the values (17%, 34%, 98%) measured by circular dichroic spectroscopy in the same nondeuterated solvent. For 3 betabellins containing a pair of β‐sheets and a related disordered peptide, their order of structural stability (12D > 12S > 14D > 14S) shown by their deuterium exchange rates in 10% CD~3~OD/0.5% CD~3~CO~2~D (pD 3.8) as measured by ESI‐MS was the same as their order of structural stability to unfolding with increasing temperature or guanidinium chloride concentration as measured by circular dichroic spectroscopy in water. Compared to monitoring deuterium exchange by proton NMR spectrometry, monitoring deuterium exchange by ESI‐MS requires much less sample (1–50 μg), much shorter analysis time (10–90 min), and no chemical quenching of the exchange reaction.