Differential Ion Mobility Separations of D/L Peptide Epimers

Life was originally assumed to utilize the L-amino acids only. Since 1980s, the D-amino acid-containing peptides (DAACPs) were detected in animals, often at extremely low levels with tremendous functional specificity. As the unguided proteomic algorithms based on peptide masses are oblivious to DAACPs, many more are believed to be hidden in organisms and novel methods to tackle DAACPs are sought. Linear ion mobility spectrometry (IMS) can distinguish and characterize the D/L epimers but is restricted by poor orthogonality to MS as in other contexts. We now bring to this area the newer technique of differential IMS (FAIMS). The orthogonality of MS to high-resolution FAIMS exceeded that to linear IMS by 6X, the greatest factor found for biomolecules so far. Hence, FAIMS has achieved the 2.5X resolution of trapped IMS on average despite a lower resolving power, fully separating all 18 pairs of representative epimer species with masses of similar to 400-5,000 Da and charge states of 1-6. A constant isomer resolution over these ranges allows projecting success for yet larger DAACPs.

Automated summary

Since the 1980s, D-amino acid-containing peptides (DAACPs) have been discovered in animals, showing tremendous functional specificity at extremely low levels. The introduction of the new technique differential IMS (FAIMS) has successfully separated all 18 pairs of epimer species, showcasing a 2.5X resolution increase compared to trapped IMS.