TY - JOUR
T1 - Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells
AU - Moully, Elamar Hakim
AU - Berns, Eric J.
AU - Mrksich, Milan
N1 - Funding Information:
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T32GM105538 and the National Cancer Institute of the National Institutes of Health under award number U54CA199091. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/10/15
Y1 - 2019/10/15
N2 - Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation - during which the PTP enzymes act on the peptide substrate - the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.
AB - Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation - during which the PTP enzymes act on the peptide substrate - the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.
UR - http://www.scopus.com/inward/record.url?scp=85072927517&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85072927517&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.9b03640
DO - 10.1021/acs.analchem.9b03640
M3 - Article
C2 - 31536703
AN - SCOPUS:85072927517
VL - 91
SP - 13206
EP - 13212
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 20
ER -