The authors thank Masae Ohno and Kazuya Nishimura for experimental assistance and advice. This work was supported by PRESTO (JPMJPR15F7), Japan Science and Technology Agency, Grants-in-aid for Young Scientists (A) (24687022), Challenging Exploratory Research (26650055), and Scientific Research on Innovative Areas (23115005), Japan Society for the Promotion of Science, and by grants from the Takeda Science Foundation and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. S.L. acknowledges support from the RIKEN International Program Associate (IPA) program.

1. Cell preparation
<ol>
	<li>Cultivate HeLa cells in a 10 cm dish at 37 &#176;C under 5% CO2 in Dulbecco&#8217;s modified Eagle&#8217;s medium containing 10% fetal bovine serum.</li>
	<li>Collect exponentially growing cells once they have reached 70% confluency, following ATCC instructions17.
	<ol>
		<li>Rinse cells with 5 mL of 1x phosphate buffered saline (PBS) (pH 7.4). Remove PBS.</li>
		<li>Add 1 mL of 0.1% trypsin-ethylenediaminetetraacetic acid (EDTA). Incubate 5 min at 37 &#1...

<ol>
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The authors declare the following competing financial interest(s): RIKEN has filed a patent application on these results with S.L. and Y.T. named as co-inventors.

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...

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 ionization1,2,3. 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 molecules in the analyzed cells, followed by electrophoretic separation and detection and quantification of each protein species. To achieve the required non-specific protein labeling, one strategy is to use fluorescent dyes that can bind to proteins via electrostatic and hydrophobic interactions, such as Coomassie Blue and Sypro-Ruby4,5,6. An alternative strategy is to use covalent labeling with dyes containing N-hydroxysuccinimide (NHS) ester or maleimide, which can covalently bind to proteins through common residues such as primary amines and thiols, respectively7,8.
Meanwhile, the sensitivity of fluorescence detection is ideal for analyzing low-abundance proteins and small numbers of cells. Single molecule fluorescence imaging is one of the most sensitive methods that allows the detection of individual fluorescent dyes and labeled proteins in vitro and in vivo9,10,11,12,13,14,15. Application of this imaging method to electrophoresis-based proteome analysis is expected to enable highly sensitive and quantitative assays by counting individual fluorescently-labeled proteins. However, it remains unclear whether labeling with fluorescent dyes is homogeneous enough across all the protein molecules, and how this homogeneity is affected by different protein species (Figure 1). Simple bulk solution measurements can be used to obtain a molar ratio of fluorescent dyes to proteins called &#8216;coupling efficiency&#8217;8 or &#8216;labeling efficiency&#8217;, but this property does not provide information on the homogeneity of the labeling among protein molecules.
Here, we describe the protocol for an assay to investigate the labeling homogeneity for all protein species in the cell (Figure 2)16. The two key steps of this assay are protein purification and imaging. In the first step, all the proteins in the cells are fluorescently labeled and biotinylated, then extracted separately using gel electrophoresis followed by electroelution. In the second step, fluorescence properties of individual protein molecules in the extracted samples are evaluated based on single molecule imaging. From this data, parameters important for the counting analysis, such as the percentage of proteins labeled with a least one dye, which we call labeling occupancy (LO)16, and the average number of fluorescent dyes bound to a single protein molecule (n&#772;dye), can be characterized. In the protocol, an optimized procedure for labeling the HeLa cell proteome with NHS ester-based Cyanine 3 (Cy3) dye is presented as an example, and can be modified with other labeling procedures according to desired research goals.

<table border="0" cellpadding="0" cellspacing="0" style="width:637px;" width="638">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">22x22x0.15 mm coverslip</td>
			<td style="width:119px;">VWR</td>
			<td style="width:136px;">470019-004</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">488 nm Argon laser</td>
			<td style="width:119px;">Coherent</td>
			<td style="width:136px;">Innova 70C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">488 nm dichroic mirror</td>
			<td style="width:119px;">Semrock</td>
			<td style="width:136px;">FF495-Di03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">488 nm emission filter</td>
			<td style="width:119px;">Semrock</td>
			<td style="width:136px;">FF02-520/28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">560 nm dichroic filter</td>
			<td style="width:119px;">Semrock</td>
			<td style="width:136px;">Di02-R561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">560 nm emission filter</td>
			<td style="width:119px;">Semrock</td>
			<td style="width:136px;">FF02-617/73</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">560 nm fiber laser</td>
			<td style="width:119px;">MBP Communications</td>
			<td style="width:136px;">F-04306-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">60x oil immersion lens</td>
			<td style="width:119px;">Olympus</td>
			<td style="width:136px;">PLAPON 60x</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Avidin</td>
			<td style="width:119px;">Nacalai-tesque</td>
			<td style="width:136px;">03553-64</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotin-PEG-amine</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:136px;">21346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biotinylated Alexa Fluor 488</td>
			<td style="width:119px;">Nanocs</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Borate</td>
			<td style="width:119px;">Nacalai-tesque</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:136px;">A9547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CHAPS</td>
			<td style="width:119px;">Dojindo</td>
			<td style="width:136px;">C008-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cy3 NHS-ester dye</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:136px;">PA13101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dialyzer&#160; - D-tube, 6-8 kDa</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:136px;">71507-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
			<td style="width:119px;">Nacalai-tesque</td>
			<td style="width:136px;">14112-94</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDC</td>
			<td style="width:119px;">Nacalai-tesque</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EMCCD camera</td>
			<td style="width:119px;">Andor</td>
			<td style="width:136px;">IiXon 897</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epi fluorescence microscope</td>
			<td style="width:119px;">Olympus</td>
			<td style="width:136px;">IX81</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Gel viewer</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:136px;">ImageQuant LAS 4000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin, streptomycine and Amphoterecin mix</td>
			<td style="width:119px;">Gibco</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasma cleaner</td>
			<td style="width:119px;">Diener Electronic</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:119px;">Wako</td>
			<td style="width:136px;">NC0792960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Size purification column 10K</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:136px;">UFC5010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:136px;">P9416</td>
			<td></td>
		</tr>
	</tbody>
</table>

proteome-wide quantification of labeling homogeneity at the single molecule level

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 &#8216;labeling occupancy&#8217; (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.

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&#8211;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 posi...

Watch this Scientific Journal Video about Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level at JoVE.com

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

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

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

This method demonstrates the calibration of quantifying protein amount in liquid sample with single-molecule process for counting. Specifically, it can illustrate how protein populations can be fluorescently labeled at each molecule level. Previous methods performed bulk measurements to analyze molar ratios of fluorescent and protein molecules, but they cannot reveal how heterogeneously protein populations are actually labeled.Our methods can do it directly at the single-molecule level. Our method can be extended towards ultrasensitive and advanced molecular concentration assays, which will lead to future diagnostic methods, allowing efficient findings of pathogenic molecules in specimens. Our method can be applied to single protein species analysis using fluorescent antibodies, as well as multiple species analysis using non-specific labels for all the proteins separated by electrophoresis or chromatography.The most critical point of the technique is obtaining data reproducibility. I recommend keeping the same experimental condition as much as possible when measuring multiple samples. Visual demonstration of this technique will provide how to make each experimental step stable and reproducible.It will also show some uncommon step in biochemistry protocols, such as the spin-coating and washing of microscope coverslips. To begin this procedure, prepare the lysing buffer and 1%Tween-20 as outlined in the text protocol. Use sodium hydroxide to adjust the pH to 12.Mix 100 microliters of lysing buffer with one microliter of one molar DDT and four microliters of 50%CHAPS. Add one microliter of the prepared cell culture and homogenize the solution by slow pipetting to avoid bubbles. Incubate in the dark at room temperature for five minutes with gentle agitation.Then, rehomogenize the solution by pipetting it up and down slowly. Add one microgram of Cy3 NHS ester dye and homogenize the solution by slowing pipetting to avoid bubbles. Incubate in the dark at room temperature for 10 minutes with gentle agitation.Repeat this process, adding the same amount of dye and incubating the sample one time. Add 100 microliters of 0.8 molar HEPES-sodium hydroxide to adjust the pH of the solution to 7.2 and to quench the reaction. Slowly pipette up and down to homogenize the solution.Next, add 20 microliters of Biotin-2-amine and five microliters of EDC. Homogenize the solution by pipetting up and down slowly. Incubate in the dark at room temperature for one hour with gentle agitation.After this, use an ultrafiltration column with a 10 kiloDalton cutoff to remove any unreacted labeling reagents and concentrate the sample. After performing standard SDS-PAGE for the protein sample and a molecular ladder, image the gel to identify the location of the protein bands. Use a sharp blade to cut out the gel portions that include protein fractions of interest based on the bands'locations.First, set out coverslips that are 22 millimeters by 22 millimeters and are 0.15 millimeters thick. Use a plasma cleaner to expose the coverslips to air plasma for one minute to clean and activate their surfaces. Next, use a spin coater to spin coat the coverslips with 200 microliters of avadin buffer for five seconds at 500RPM, followed by 30 seconds at 1000RPM.Incubate the coverslips at room temperature for 15 minutes to let them air dry. To prepare coverslips for the protein sample, use a protein sample that has been diluted. Place 100 microliters of the diluted sample onto the center of the avadin-coated coverslips.Incubate in the dark at room temperature for 15 minutes. To prepare the positive control, place 100 microliters of purified fluorescent biotin in five micromolar HEPES-sodium hydroxide at pH 7.2 in the center of an avadin-coated coverslip. Incubate in the dark at room temperature for 15 minutes.To prepare the negative control, spin coat plasma coverslips with 200 microliters of five HEPES-sodium hydroxide with two milligrams per milliliter of BSA without avadin. Add 100 microliters to purified fluorescent biotin in five micromolar HEPES-sodium hydroxide at pH 7.2. Incubate in the dark for 15 minutes at room temperature.After this, rinse each coverslip with 200 microliters of distilled water by pipetting at the edges, making sure to not touch the middle of the coverslip with the pipette tip. Repeat this wash three times. Then, expose an equal number of new coverslips to air plasma for one minute.Place a cleaned coverslip on top of each sample-bound coverslip to avoid drying. Start up a wide-field or evanescent field fluorescence microscope. Set a coverslip onto the microscope and find the focus.Then, perform a tile scan to obtain at least 100 images. In this study, the labeling homogeneity of each labeled protein species in cells is quantified after separation with SDS-PAGE. The raw image data for different molecular weight fractions of proteins from HeLa cell lysate, as well as the positive and negative controls, are seen here.While both the protein sample and the positive control exhibit between 100 and 500 spots per image, the negative control exhibits very few to none. This shows that the process sufficiently inhibits non-specific binding of dyes to the coverslip surface. Spot intensity histograms for the protein samples and the positive control present multiple peaks, which represent the stochastic binding of dyes to primary amines in proteins and avadin tetramer structures respectively.All spots showed blinking and stepwise photobleaching with continuous laser excitation, highlighting observation at the single molecule level. The labeling occupancy, or LO, is then calculated from the ratio of the number of spots in every protein sample to that in the positive control. The LO measured from the HeLa cell lysate sample ranges from 50%for the smaller molecular weight fraction, to 90%for the higher molecular weight fraction.The LO for the whole proteome sample without separation is seen to be 72%It is critical to use freshly made reagents and to avoid any dust, especially during the preparation of the coverslips. Following this procedure, single molecule counting analysis in a certain volume can be performed to quantify each protein's concentrations. For example, analysis on any raw product, or gel and capillary electrophoresis products, can be performed.This technique paves the way for utilizing single molecule fluorescence imaging to molecular medicine, organic chemistry, and omics analysis. By bridging the concept between concentration and numbers. Concentrated sodium hydroxide is corrosive, and adequate protection should be worn when handling it.Also, high power laser radiation can cause eye damage, so protective goggles may be worn.

proteome-wide-quantification-labeling-homogeneity-at-single-molecule

Research

JoVE Journal

Biochemistry

6.0K visualizações.  RIKEN. Este método demonstra a calibração da quantidade de proteína quantificada em amostra líquida com processo de molécula única para contagem. Especificamente, pode ilustrar como as populações de proteínas podem ser rotuladas fluorescentemente em cada nível de molécula. Métodos anteriores realizaram medições em massa para analisar as proporções molares de moléculas fluorescentes e proteicas, mas não podem revelar como populações heterogêneas de proteínas são realmente rotu...