Investigations of the Mechanism of the “Proline Effect” in Tandem Mass Spectrometry Experiments: The “Pipecolic Acid Effect”

Abstract

The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established "proline effect" through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give yn (+) ions. In contrast, Pip- and NMA-fragment through a different mechanism, the "pipecolic acid effect," selectively at the amide bond C-terminal to the Pip/NMA residue to give bn (+) ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the "proline effect' can in part be rationalized by the increased basicity of the prolyl-amide site, the "pipecolic acid effect" cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the "pipecolic acid effect" is stronger than the "proline effect."

Top-down mass spectrometry of hybrid materials with hydrophobic peptide and hydrophilic or hydrophobic polymer blocks

“…1b, agrees well with the sequence XVPGVGVPGVGX, comprising an acetylated propargyl glycine at the N-terminus and an amidated propargyl glycine at the C-terminus. 70 The sodium ion affinities of proline (196 kJ mol −1 ), 68 valine (173 kJ mol −1 ) 68 and glycine (161 kJ mol −1 ) 68 follow the same order as the corresponding proton affinities (921, 911 and 887 kJ mol −1 , respectively 71 ). According to established peptide ion fragmentation mechanisms, [67][68][69] these fragments are generated by rearrangement fragmentations at the C(vO)-NH bonds which break the peptide into two segments, one containing the N-terminus and the other the C-terminus.…”

Analytical chemistry research at primarily undergraduate institutions: training tomorrow's investigators

“…In 2012, Hughey and two undergraduate students published an Analytical Chemistry paper that investigated the role of polar protic and aprotic solvents on negative-ion ESI. 52 Another group used LC-MS-MS with isobaric tags to measure metabolic changes in diabetic mice, 53 and a third group developed data acquisition and processing methods to interpret MALDI data into quantitative measures of the phosphoproteome. Another strategy at PUIs is to engage in collaborative MS research.…”

Infrared Multiple-Photon Dissociation Action Spectroscopy of the b2+ Ion from PPG: Evidence of Third Residue Affecting b2+ Fragment Structure

“…[12,13] Consequently, proline has been part of many peptide fragmentation model studies. Proline-containing peptides have been shown to have a higher propensity for forming cis peptide bonds at the Pro residue as its barrier to trans/cis isomerization of the N-terminally adjacent peptide bond is ~55 kJ/mol compared to the average ~80 kJ/mol for the other 19 protein amino acids.…”

“…[13,17–20] In analysis of precursor peptides containing proline, Masson et al, using both spectroscopy and ion mobility, confirmed that GPGG was comprised of a larger kinetically trapped trans -Pro population and a smaller cis -Pro population that was found to be more thermodynamically stable in the gas-phase. [21]…”