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

Abstract

Despite 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 co-eluting 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 inter-isomeric 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.

Boosting basic-peptide separation through dynamic electrostatic-repulsion reversed-phase (d-ERRP) liquid chromatography

“…S4 and Table S3 ESI †). Notably, by using TBAHSO 4 as an additive, we noted the inversion of the elution order between [D-His] 1 -GLUC and [Glu] 3 -GLUC, and [Glu] 24 -GLUC and [Glu] 20 -GLUC. Moreover, a higher content of ACN (compared to that used in the last two analyses described above) is needed in order to elute analytes.…”

“…Indeed, over the years, several analytical techniques have been developed ad hoc for recognizing and describing peptide stereochemistry, such as enantioselective liquid chromatography, 19,20 NMR, 21 selective enzymatic digestion, 22 and some exciting applications involving highly specic mass spectrometry techniques. 23,24 Particularly for epimers, it is also possible to use reversedphase chromatography (RPC). RPC is one of the most versatile and exploited chromatographic technique, it is used in the analysis of a wide range of compounds, and even if it cannot provide separation of enantiomers, it allows separating diastereomers such as peptide epimers.…”

“…31 This peptide is made up of 29 L-amino acids, and it is extremely challenging from an analytical point of view because of the different modications arising during its synthesis, such as racemization of the rst histidine, that generate an epimeric isobaric impurity ([D-His] 1 -GLUC 2). Moreover, it can undergo deamidation of glutamines in positions 3, 20, and 24, producing the impurities named [Glu] 3 -GLUC 3, [Glu] 20 -GLUC 5 and [Glu] 24 -GLUC 6 and asparagines, forming [Asp] 28 -GLUC 4 (Fig. 1).…”

General lack of structural characterization of chemically synthesized long peptides

“…If experimental parameters are selected carefully, the gas phase CCS can provide reliable information on the conformation in the liquid phase. For example, IM‐MS allowed the characterization of peptide−synthetic polymer conjugates, the study of conformer preferences for hydrophobic antimicrobial peptides, the identification of lasso peptide topologies, and the separation of d ‐amino‐acid‐containing peptides . IM‐MS can even be coupled with 1D or 2D LC as shown for the characterization of antibody–drug conjugates, in which initial separation by size is achieved by hydrophobic interaction chromatography.…”

Acid‐based approach for separation of peptide epimers using IM‐MS

“…This factor, in turn, is strictly related to the type of ion mobility drift tube that is used. For example, recently reported studies underlined the resolution advantage of the custom trap ion mobility drift tube (TIMS) over the widely available, but more poorly resolving, traveling‐wave IMS (TWIMS) implemented in the Synapt G2 instrument (Waters, Milford, MA), which was 5 to 10‐fold less powerful . Temperature, drift gas composition, and pressure are other variables that affect separation, as well as electric field parameters and conformational flexibility of analyzed ions …”