Mass Spectrometric Differentiation of Linear Peptides Composed of l-Amino Acids from Isomers Containing One d-Amino Acid Residue

Abstract

MS/MS of electrosprayed ions is shown to have the capacity to discriminate between peptides that differ by configuration about their alpha-carbons. It is not necessary for the peptides to possess tertiary structures that are affected by stereochemistry, since five epimers of the pentapeptide, H2N-Gly-Leu-Ser-Phe-Ala-OH (GLSFA) all display different collisionally activated dissociation (CAD) patterns of their protonated parent ions. The figure of merit, r, is a ratio of ratios of fragment ion abundances between stereoisomers, where r = 1 corresponds to no stereochemical effect. Values of r as high as 3.8 are seen for diastereomer pairs. Stereochemical effects are also seen for the diprotonated dodecapeptide H2N-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-OH (LVFFAEDVGSNK), a tryptic fragment from the amyloid beta-protein. Triply charged complexes of the protonated dodecapeptide with cobalt(II) ions undergo CAD at lower collision energies than do doubly protonated LVFFAEDVGSNK ions. Statistically significant (p < 0.01) differences between the all-L-dodecapeptide and the ones containing a d-serine or a D-aspartic acid are observed.

Characterization of GdFFD, a d-Amino Acid-containing Neuropeptide That Functions as an Extrinsic Modulator of the Aplysia Feeding Circuit

“…Bonferroni post hoc test is as follows: **, p Ͻ 0.01; ***, p Ͻ 0.001. experiment, and this difference can be manifested as different branching ratios among product ions. It has been shown that several different MS techniques can be used to probe this thermochemical difference between diastereomers (34,61,62,(72)(73)(74). We have also shown that simultaneous identification and characterization of potential DAACPs by MALDI-TOF/ TOF MS can be applied to distinguish D-isomer peptides in biological samples as small as individual neurons (38).…”

“…Homology searches that exploited structural and functional similarities between prohormone sequences in other species (75) allowed us to generate a hypothesis for the existence of two Aplysia putative DAACPs encoding the prohormones GdFFD and YdAEFLa. Prior to MS characterization, we confirmed the expression and distribution of these prohormones in the CNS by molecular techniques and investigated their final gene products in isolated neurons using proven MALDI-TOF/TOF MS. We took advantage of the distinct fragmentation patterns that can arise between peptide epimers using both MALDI MS/MS-and LC-ESI MS/MSbased approaches (34,38,61,73,74) to distinguish DAACPs and their all L-peptide counterparts. Our identification of GdFFD in Aplysia, similar to achatin-I (GdFAD) in Achatina, validates our homology-based MS approach as being effective for identifying novel DAACPs in related animals.…”

Use of synthetic analogues in confirmation of structure of the peptide antibiotics Maltacines

“…Both electron capture dissociation (ECD) and CID have been used in the study of fragmentation of peptides containing damino acid substitutions [14,15]. According to these studies, it is the doubly charged protonated parent ions that exhibit the highest stereo chemical sensitivity [14].…”

Experimental strategies for the analysis of d-amino acid containing peptides in crustaceans: A review

“…This was made possible because the presence of a d-residue induces a difference in the fragmentation pattern upon analysis [23]. Until our work described in the next paragraph, this phenomenon has been mostly analyzed after ionization of peptides by electrospray, such as in [24].…”