Single amino acid resolution of proteolytic fragments generated in individual cells.
The complex nature of enzyme regulation mandates that enzyme activity profiles be measured in the context of the intact cell. Single-cell capillary electrophoresis (CE) coupled with laser-induced fluorescence is a powerful approach for quantitation and separation of analytes present in small samples and single live cells; however, it does not allow for the definitive identification of the reaction products. On the other hand, mass spectrometry (MS) is able to identify analytes but still lacks the requisite sensitivity for most single-cell analysis applications. Thus, it follows that by determining the relative amounts of reaction products generated in single cells using CE and by producing larger quantities of these products using bulk cell populations to identify them using MS, it is possible to determine enzyme activity profiles in single cells. In this study, the applicability of this approach was demonstrated by examining the intracellular fate of a protease substrate derived from the beta-amyloid precursor protein (beta-APP). In single live TF-1 cells, three distinct fragments were generated from the beta-APP peptide, which differed by a single uncharged amino acid. The CE measurements indicated that the proteolytic fragment profiles (i.e., the relative amounts of each fragment) were consistent from cell to cell but that they were different from those obtained in cell lysates. Furthermore, measurements obtained at the single cell level made it possible to observe a modest but statistically significant negative correlation between the total amount of beta-APP peptide loaded in cells and the fraction of peptide that remained intact. This study demonstrates how single-cell CE, MS, and peptide substrates can be combined to identify and measure enzyme activities in single live cells.
Brown
Robert B
RB
Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario, Canada.
Hewel
Johannes A
JA
Emili
Andrew
A
Audet
Julie
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002470:Cell Survival; D002477:Cells / Q000378:metabolism*; D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational
D000596:Amino Acids; D016564:Amyloid beta-Protein Precursor; D002457:Cell Extracts; D010446:Peptide Fragments