Analysis of Peptide Stereochemistry in Single Cells by Capillary Electrophoresis–Trapped Ion Mobility Spectrometry Mass Spectrometry

Abstract

Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic products. Here, we applied mass analysis of single neurons to investigate cell–cell signaling peptides. The multiplicity of endogenous cell–cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE–TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

“…Stereochemical configuration of three neuropeptides derived from the pleurin precursor in individual neurons isolated from the central nervous system of Aplysia californica was determined by CE coupled to trapped ion mobility spectrometry MS (CE‐TIMS‐MS) [274]. Analysis of the mobility profiles has shown that more than 98% of the pleurin‐derived peptides occur in nonisomerized, all‐ l forms in the individual neuron cells but 44% of the Plrn2 peptide was found to be present in the isomerized, d ‐residue‐containing form in the nerve tissue.…”

What the Microscale Systems “See” In Biological Assemblies: Cells and Viruses?

“…Because of its specific advantages, including low reagent consumption, desirable separation efficiency, and high sensitivity, CE–MS is a promising technique for the single-cell assay. − FePt NPs can significantly suppress tumor cell proliferation . Therefore, it is necessary to study the cytotoxicity mechanism and cellular distribution of FePt NPs.…”

Comprehensive Online Reversed-Phase × Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid Analysis

“…They are also present in (natural, synthetic, and therapeutic) peptides, requiring their analytical determination as part of structure elucidation and/or quality control . A direct enantioresolution of underivatized and derivatized AAs using chiral stationary phases is nowadays the first choice. − Common examples for achiral precolumn derivatization supporting chiral separation comprise 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC), , 1-fluoro-2,4-dinitrobenzene (DNB-F, Sanger’s reagent), 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD), dansyl chloride (Dns), , and 9-fluorenylmethyl-chloroformate (Fmoc-Cl). − These reagents commonly improve the retention and enantioselective behavior, , introduce chromophoric and fluorophoric detectability, can enhance detection sensitivity of AAs with poor electrospray ionization efficiency, and deliver signature ions (in MS/MS detection). , Chiral stationary phases, the workhorses for direct HPLC enantiomer separation, have often remarkable enantioselectivity, but suffer from limited chemoselectivity and lower efficiencies (compared to highly efficient RP congeners). , Hence, the combination of the two principles is quite obvious and should lead to an enhanced separation technology platform.…”