A unique collision-induced dissociation pattern was observed for protonated polyproline peptides of length n in which y nϪ2 and/or y nϪ4 ions were formed in much higher abundance than any other product ions. Cleavage occurs only at every other amide bond, such that product ions are formed only from the losses of even numbers of proline residues. Exclusive losses of even numbers of proline residues were not observed from sodiated peptides. Further study of the tandem mass spectrometry (MS/MS) patterns of protonated proline-rich peptides showed that the substitution of alanine in the second position of polyproline peptides did not prevent the dominant formation of y nϪ2 and y nϪ4 ions. The loss of ProAla to form the y 8 ion from (ProAlaPro 8 NH 2 ϩH) ϩ was as abundant as the loss of ProPro from (Pro 10 NH 2 ϩH) ϩ . However, modification of the peptides that presumably affected the location of the proton on the peptide did alter the MS/MS spectra. Pro 10 and Pro 5 with blocked N-termini or with arginine substituted for the first proline residue did not form abundant y nϪ2 or y nϪ4 ions. MS 3 and double resonance experiments showed that dissociation of intermediate y n product ions can produce y nϪ2 ions, but are not necessary dissociation pathway intermediates. This analysis suggests that the ionizing proton must be located at the N-terminus for the peptide ion to dissociate in this manner. (J Am Soc Mass Spectrom 2007, 18, 2198 -2203) © 2007 American Society for Mass Spectrometry P roline (Pro) is the only naturally occurring amino acid that is bonded to the nitrogen atom of the peptide backbone. The absence of the amide hydrogen prevents polyproline peptides from forming alpha helices. Instead, two possible helical structures of polyproline can be found in solution, named ProI and ProII. ProI exists in solution as a right-handed helix with 3.3 residues per turn, and all peptide bonds are cis bonds. ProII is a left-handed helix with 3.0 residues per turn, and all peptide bonds are trans bonds. The type of helix formed varies with the type of solvent in which the polyproline is dissolved. ProI is found in less protic solvents such as butanol or propanol, whereas ProII exists in more protic solvents such as water. Counterman and Clemmer [1] have shown with ion mobility spectrometry and supporting computational studies that when polyproline peptides are introduced into the gas phase from propanol solutions, features of the ProI helix persist in the gas phase. This was observed for many different lengths of polyproline chains. However, electrospray ionization (ESI) of aqueous solutions did not produce ion mobility data consistent with ProII helices. It was reasoned that the elongated structure of the ProII helix could not be stabilized in the absence of solvent [1,2]. It already has been established that the solvent choice has no effect on the dissociation energetics of polyproline peptides [3]. Interestingly, a unifying feature was established for all polyproline peptides examined, regardless of the original solvent....