Differential Ion Mobility Separations of d/l Peptide Epimers

Abstract

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 6×, the greatest factor found for biomolecules so far. Hence, FAIMS has achieved the 2.5× resolution of trapped IMS on average despite a lower resolving power, fully separating all 18 pairs of representative epimer species with masses of ∼400–5,000 Da and charge states of 1–6. A constant isomer resolution over these ranges allows projecting success for yet larger DAACPs.

Ion Mobility Spectrometry of Superheated Macromolecules at Electric Fields up to 500 Td

“…3,33,35,36 Hence, MS is more orthogonal to FAIMS than to linear IMS, typically by 3−6-fold for lipids and peptides. 24,26 Thus, FAIMS can disentangle isomers better than linear IMS of equal resolving power (R), that is, FAIMS with R = 100 may compete with IMS with 24 R > 300. Indeed, AP-FAIMS has broadly separated the regioisomers of aromatic compounds, 36,37 anomers, and linkage or position isomers of saccharides, 38 lipids with swapped acyl chains or double bond locations/symmetries, 26,27 peptides with sequence inversions, D-amino acids, 24 or alternative post-translational modification sites, 22 and even isotopomers.…”

“…24,26 Thus, FAIMS can disentangle isomers better than linear IMS of equal resolving power (R), that is, FAIMS with R = 100 may compete with IMS with 24 R > 300. Indeed, AP-FAIMS has broadly separated the regioisomers of aromatic compounds, 36,37 anomers, and linkage or position isomers of saccharides, 38 lipids with swapped acyl chains or double bond locations/symmetries, 26,27 peptides with sequence inversions, D-amino acids, 24 or alternative post-translational modification sites, 22 and even isotopomers. 39 Multiple isomers were resolved for proteins, some (perhaps, "protomers" with distinct protonated sites) within the highly charged unfolded conformers co-eluting in linear IMS.…”

“…Both linear IMS and FAIMS, coupled to ESI-MS, are now a staple of bioanalysis . The headline applications have been the fractionation of proteomic or metabolomic samples before the MS step and identification of isomeric peptides ,− and lipids. − With MS expanding to ever heavier ions, a growing area is the separation and conformational elucidation of macromolecules such as proteins and their complexes. − ,− …”

“…An inherent drawback of linear IMS is the lacking orthogonality to MS, due to the relationship between the ion mass ( m ) and Ω avg (especially within a chemical class). − This causes inefficient utilization of the 2-D IMS–MS space and limited isomer resolution. The correlation between m and Δ K is much looser, for one Δ K (unlike K ) can have either sign. ,,, Hence, MS is more orthogonal to FAIMS than to linear IMS, typically by 3–6-fold for lipids and peptides. , Thus, FAIMS can disentangle isomers better than linear IMS of equal resolving power ( R ), that is, FAIMS with R = 100 may compete with IMS with R > 300. Indeed, AP-FAIMS has broadly separated the regioisomers of aromatic compounds, , anomers, and linkage or position isomers of saccharides, lipids with swapped acyl chains or double bond locations/symmetries, , peptides with sequence inversions, d -amino acids, or alternative post-translational modification sites, and even isotopomers .…”

An atmospheric pressure ion funnel with a slit entrance for enhancing signal and resolution in high resolution differential ion mobility mass spectrometry

“…Owing to the use of relatively long planar electrodes (∼65 mm), wide analytical gap widths (∼2 mm), and low molecular weight carrier gases, the performance of DMS in terms of resolving power and separation capacity has been significantly improved. For example, high resolution DMS can separate diastereomers, 12,13 isomeric perfluoroalkyl substances, 14 disaccharide epimers, 15 peptide isomers, 16,17 isomeric lipids, 18 and protein ion protonation isomers with the same mass, charge and collision cross section. 19 Moreover, resolving power values as high as ∼500 have been reported for high resolution DMS.…”