Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Summary

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Abstract

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed ‘labeling occupancy’ (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Introduction

Proteome analysis, which aims to quantify the entire set of protein molecules expressed in the cell, is a valuable approach in current biological and medicinal studies. This analysis commonly relies on mass spectrometry, which identifies protein species based on spectra generated through protein ionization^1,2,3. An alternative method for proteome analysis is electrophoresis, including polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis and two-dimensional (2D) gel electrophoresis. This method relies on non-specific fluorescent labeling of all the protein mole....

Protocol

1. Cell preparation

1. Cultivate HeLa cells in a 10 cm dish at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum.
2. Collect exponentially growing cells once they have reached 70% confluency, following ATCC instructions¹⁷.
   1. Rinse cells with 5 mL of 1x phosphate buffered saline (PBS) (pH 7.4). Remove PBS.
   2. Add 1 mL of 0.1% trypsin-ethylenediaminetetraacetic acid (EDTA). Incubate 5 min at 37 °C.
   3. Monitor the progression of cell dissociation by microscopy.
   4. Once cells become round and are detached from the dish, add 4 ....

Results

Figure 4 represents raw image data for different molecular weight fractions of proteins from HeLa cell lysate, as well as the positive and negative control. While both the protein sample and positive control exhibit 100–500 spots per image, the negative control displays none or a few spots, showing that the protocol sufficiently inhibits non-specific binding of dyes to the coverslip surface. Spot intensity histograms for the protein samples and the positive c.......

Discussion

This paper describes a protocol to quantify the labeling homogeneity of each labeled protein species in cells after separation with SDS-PAGE (step 3). The separation method can be substituted with other methods such as liquid chromatography or capillary electrophoresis, which allow separation and fractionation of cellular proteins with high resolution, while requiring special equipment²³. The labeling method using NHS-ester in the current protocol can be replaced with other methods using maleimide.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
22x22x0.15 mm coverslip	VWR	470019-004
488 nm Argon laser	Coherent	Innova 70C
488 nm dichroic mirror	Semrock	FF495-Di03
488 nm emission filter	Semrock	FF02-520/28
560 nm dichroic filter	Semrock	Di02-R561
560 nm emission filter	Semrock	FF02-617/73
560 nm fiber laser	MBP Communications	F-04306-2
60x oil immersion lens	Olympus	PLAPON 60x
Avidin	Nacalai-tesque	03553-64
Biotin-PEG-amine	Thermo Scientific	21346
Biotinylated Alexa Fluor 488	Nanocs
Borate	Nacalai-tesque
BSA	Sigma-Aldrich	A9547
CHAPS	Dojindo	C008-10
Cy3 NHS-ester dye	GE Healthcare	PA13101
Dialyzer  - D-tube, 6-8 kDa	Merck Millipore	71507-M
DMEM	Sigma-Aldrich
DTT	Nacalai-tesque	14112-94
EDC	Nacalai-tesque
EMCCD camera	Andor	IiXon 897
Epi fluorescence microscope	Olympus	IX81
Gel viewer	GE Healthcare	ImageQuant LAS 4000
Penicillin, streptomycine and Amphoterecin mix	Gibco
Plasma cleaner	Diener Electronic
SDS	Wako	NC0792960
Size purification column 10K	Merck Millipore	UFC5010
Tween 20	Sigma-Aldrich	P9416