ac7b03401_si_001.pdf (1.28 MB)
Fast and Effective Ion Mobility–Mass Spectrometry Separation of d‑Amino-Acid-Containing Peptides
journal contribution
posted on 2017-10-05, 00:00 authored by Kevin Jeanne Dit Fouque, Alyssa Garabedian, Jacob Porter, Matthew Baird, Xueqin Pang, Todd D. Williams, Lingjun Li, Alexandre Shvartsburg, Francisco Fernandez-LimaDespite often minute
concentrations in vivo, d-amino acid
containing peptides (DAACPs) are crucial to many life processes. Standard
proteomics protocols fail to detect them as d/l substitutions
do not affect the peptide parent and fragment masses. The differences
in fragment yields are often limited, obstructing the investigations
of important but low abundance epimers in isomeric mixtures. Separation
of d/l-peptides using ion mobility spectrometry (IMS) was
impeded by small collision cross section differences (commonly ∼1%).
Here, broad baseline separation of DAACPs with up to ∼30 residues
employing trapped IMS with resolving power up to ∼340, followed
by time-of-flight mass spectrometry is demonstrated. The d/l-pairs coeluting in one charge state were resolved in another, and
epimers merged as protonated species were resolved upon metalation,
effectively turning the charge state and cationization mode into extra
separation dimensions. Linear quantification down to 0.25% proved
the utility of high resolution IMS-MS for real samples with large
interisomeric dynamic range. Very close relative mobilities found
for DAACP pairs using traveling-wave IMS (TWIMS) with different ion
sources and faster IMS separations showed the transferability of results
across IMS platforms. Fragmentation of epimers can enhance their identification
and further improve detection and quantification limits, and we demonstrate
the advantages of online mobility separated collision-induced dissociation
(CID) followed by high resolution mass spectrometry (TIMS-CID-MS)
for epimer analysis.