Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells

Abstract

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.

Figure 1.

Overview of the single-cell SAMDI assay. A suspension of individual cells, prepared in a buffer containing HRP, is sorted into a low-volume, 384-well plate by flow cytometry. Each well already contains lysis buffer and a phosphopeptide substrate (Ac-IpYERC-NH₂). Once deposited, the cells are lysed, and active PTP enzymes can act on the substrate. Following an incubation period, the contents of the well plate are split into two pools. One pool is transferred onto an array of 384 gold islands—each presenting a maleimide-functionalized monolayer—to immobilize both substrate and product present in solution. The array is analyzed using SAMDI mass spectrometry, which quantitates PTP activity in each well by reporting the amount of substrate and product and therefore the yield. To the second pool, a luminescent HRP substrate is added, and the resulting luminescence signal is used to eliminate any wells that did not receive a cell due to error by flow cytometry (which are indicated by the ‘X’s in the heatmap). SAMDI measurements from the remaining wells reveal the distribution of single-cell activities.

Figure 2.

PTP activity measurements using SAMDI. (A) The phosphopeptide substrate and the corresponding dephosphorylated product that are present in a reaction mixture are captured onto a maleimide-presenting self-assembled monolayer. (B) Representative SAMDI spectra from wells containing 0 cells (top) and 1 cell (bottom). Blue star: Adduct peak of substrate. Green star: Adduct peak of product.

Figure 3.

Histograms showing the distribution of PTP activities in individual cells from a population, with corresponding fits to a gamma distribution. A control experiment where no cells were added to the wells (48 wells, gray) reveals a background level of measured activity. An experiment to measure activities in individual cells reveals a broad distribution (143 single cells, blue), and a control experiment that measures activities in an identical homogenous lysate (96 wells, pink) reveals the technical variance in the assay, and demonstrates that a significant fraction of the distribution in the analysis of a cell population is due to variation in PTP activity.

Figure 4.

SAMDI analysis of the distribution of activities in a mixed population of cells. MDA-MB-231 cells (labeled with CellTracker™ Green CMFDA) and HEK293 cells (labeled with CellTracker™ Red CMPTX) were mixed into a single suspension. The top histogram shows the distribution of measurements of the background (0 cells) and a fit to a normal distribution (gray line). The green and red histograms show the distributions of single-cell activities measured from MDA-MB231 and HEK293 cells, respectively, sorted with fluorescence gating, and the lines represent gamma distribution fits.