Discriminating d-Amino Acid-Containing Peptide Epimers by Radical-Directed Dissociation Mass Spectrometry

Abstract

The presence of a single D-amino acid in a peptide is very difficult to detect. Mass spectrometry-based approaches rely on differences in fragmentation between all L-amino acid-containing peptides and single D-amino acid-containing peptides (which are epimers) for identification. The success of this approach is dependent on the structural sensitivity of the fragmentation method. Recently, experiments have demonstrated that fragmentation initiated by radical chemistry, or radical-directed dissociation (RDD), is particularly sensitive to the structure of the ion being fragmented. Herein, RDD is used to identify the presence of D-serine, D-alanine, or D-aspartic acid in eight biologically relevant peptides. It is demonstrated that chiral disambiguation by RDD is dependent on both the initial radical site and subsequent radical migration. Fortuitously, RDD can be initiated by a variety of different radical precursors which can be associated with the peptide via covalent or noncovalent means, and RDD can be examined in all observable charge states (both positive and negative). This diversity enables numerous initial radical sites and migration pathways to be explored. For all but one of the peptides that were examined, RDD provides significantly better chiral discrimination than CID. Quantitation of peptide epimers by RDD is also described.

Fast and Effective Ion Mobility–Mass Spectrometry Separation of d-Amino-Acid-Containing Peptides

“…The latter can also reduce f LOQ and f LOD by quantifying the isomer ratio at minor peaks partly covered by the wings of major peaks. In principle, an RDD analysis 28 with f LOD = 2% after IMS separation with present f LOD < 0.25% would yield total f LOD < 50 ppm (assuming sufficient signal averaging). Our current platforms allow no EC/TD or RDD, but can perform CID that provides some epimer discrimination.…”

A d-Amino Acid-Containing Neuropeptide Discovery Funnel

“…6 One way to overcome this difficulty is via the use of MS/MS-based techniques and comparing peptides with only l -amino acids to their DAACP epimers. 7,14−18 Recently, MALDI-TOF/TOF was used for the discovery of d -Leu in the peptide GH-2. 19 This study also established a stereosensitive fragmentation effect of d -amino acids from various amino acid classes on different peptide sequences, which can aid in the discovery of DAACPs through MALDI-TOF/TOF by observing the fragmentation ratios of specific peptides.…”

Investigation of Fragmentation of Tryptophan Nitrogen Radical Cation

“…While we did not investigate the structure of the resulting ion at m/z 146 in detail, a similar product was formed in the reaction of some other ions (e.g., 4-hydroxypyridinium and 2, the major fragment in CID of π-radical cation of tryptophan [7,15,34]. These data are given in Supplementary Figure S3.)…”

“…Understanding fragmentation mechanisms of peptide radical ions is crucial for interpreting experimental data in techniques like electron capture dissociation [5], electron transfer dissociation [6], and radical-directed dissociation [7]. Among the various amino acid-based radicals, tryptophan is especially intriguing as it can form two types of biologically important radicals-the tryptophan π-radical cation (Trp •+ ) and the indolyl radical (TrpN • ).…”