TY - JOUR
T1 - Analysis of Peptide Stereochemistry in Single Cells by Capillary Electrophoresis–Trapped Ion Mobility Spectrometry Mass Spectrometry
AU - Mast, David H.
AU - Liao, Hsiao Wei
AU - Romanova, Elena V.
AU - Sweedler, Jonathan V.
N1 - Funding Information:
We thank Professor T. Do at the University of Tennessee Knoxville for useful discussions about ion mobility spectrometry. We also thank C. Mullens, and T. Srikumar at Bruker Corp. for guidance and assistance on TIMS measurements and the operation of the timsTOF pro mass spectrometer. Research reported in this publication was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke by Award No. R01NS031609, and the National Institute on Drug Abuse by Award No. P30DA018310. The National Resource for Aplysia (Miami, FL) is funded by PHS Grant P40OD010952. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/4/20
Y1 - 2021/4/20
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.1c00445
DO - 10.1021/acs.analchem.1c00445
M3 - Article
C2 - 33825437
SN - 0003-2700
VL - 93
SP - 6205
EP - 6213
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 15
ER -