Name: genome-wide quantification of translation in budding yeast by ribosome profiling
Uploaded: 2017-12-21T14:00:00
Description: Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

This work was supported by the National Institutes of Health grants AG040191 and AG054566&#160;to VML. This research was conducted while VML was an AFAR Research Grant recipient from the American Federation for Aging Research.

1. Extract Preparation
<ol>
	<li>Streak yeast strains from frozen stocks for single colonies on YPD plates (1% yeast extract, 2% peptone, 2% glucose, and 2% agar). Incubate the plates at 30 &#176;C for 2 days.</li>
	<li>Inoculate yeast from a YPD plate (use a single colony) into 15 mL of YPD medium (1% yeast extract, 2% peptone, 2% glucose) in a 50 mL conical centrifuge tube and grow overnight with shaking (200-250 rpm) at 30 &#176;C.</li>
	<li>Dilute culture into 500 mL of YPD medium in a 2 L sterile flask, so that th...

<ol>
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The Ribo-Seq approach has emerged as a powerful technology for the analysis of mRNA translation in vivo at the genome-wide level 3. Studies using this approach, which allows monitoring translation with single-codon resolution, has contributed to our understanding of translational regulation. Despite its advantages, Ribo-Seq has several limitations. Ribosomal RNA (rRNA) fragments are always co-purified during isolation of ribosome-protected footprints decreasing the yield of useful sequenc...

mRNA translation is one of the fundamental processes in the cell, which plays an important role in the regulation of protein expression. Therefore, mRNA translation is tightly controlled in response to different internal and external physiological stimuli 1,2. However, the mechanisms of translational regulation remain understudied. Here, we describe the protocol for the genome-wide quantification of translation in budding yeast by ribosome profiling. The overall goal of the ribosome profiling technique is to study and quantify the translation of specific mRNAs under different cellular conditions. This technique uses next-generation sequencing to quantitatively analyze ribosome occupancy throughout the genome and allows monitoring the rate of protein synthesis in vivo at the single codon resolution 3,4. Currently, this method provides the most advanced means of measuring the levels of protein translation, and has proven to be a useful discovery tool providing information that cannot be revealed by other currently available techniques, e.g. microarrays or translation state array analysis (TSAA) 5. As ribosome profiling reports on the combined changes in transcript levels and translational output, it also provides much greater sensitivity compared to other methods.
This approach is based on deep sequencing of ribosome-protected mRNA fragments 3. During protein translation, ribosomes protect ~ 28 nt portions of the mRNA (called footprints) 6. By determining the sequence of the ribosome-protected fragments, Ribo-Seq can map the position of ribosomes on the translated mRNA and identify which regions of mRNA are likely to be actively translated into protein 3,7. In addition, we can quantitatively measure the translation of mRNA by counting the number of footprints that align to a given mRNA transcript.
In order to isolate the ribosome-protected fragments, cell lysates are initially treated with a translation inhibitor to stall the ribosomes followed by ribonuclease digestion. Whereas free mRNA and portions of translated mRNAs not protected by ribosomes are degraded by ribonuclease, the ribosome-protected mRNA fragments can be recovered by purifying intact ribosome-footprint complexes. These mRNA footprints are then converted into cDNA library and analyzed by deep sequencing (Figure 1). In parallel to ribosome profiling, intact mRNA is extracted from the same sample and sequenced. By comparing the level of translation identified by Ribo-Seq with mRNA abundance measurements, we can identify genes that are specifically up- or down-regulated at the level of translation and calculate translation efficiency of mRNA at the genome-wide level. While the protocol described in this article is specific for yeast, it should be also useful for&#160;researchers who will try to establish the Ribo-Seq protocol in other systems.

<table>
	<tbody>
		<tr>
			<td>0.45 &#956;M membrane filters</td>
			<td>Millipore</td>
			<td>HVLP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9261</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL centrifugal filters (100 kDa MWCO)</td>
			<td>Millipore</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.0</td>
			<td>Invitrogen</td>
			<td>AM9850G</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.5 (pH 8.0 at 4&#176;C)</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>10X TBE buffer</td>
			<td>Invitrogen</td>
			<td>AM9863</td>
			<td></td>
		</tr>
		<tr>
			<td>10% TBE-urea gel</td>
			<td>Invitrogen</td>
			<td>EC6875BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>15% TBE-urea gel</td>
			<td>Invitrogen</td>
			<td>EC6885BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M MgCl2</td>
			<td>RPI</td>
			<td>M24500-10.0</td>
			<td></td>
		</tr>
		<tr>
			<td>2X TBE-urea sample buffer</td>
			<td>Invitrogen</td>
			<td>LC6876</td>
			<td></td>
		</tr>
		<tr>
			<td>3M NaOAc, pH 5.5</td>
			<td>Invitrogen</td>
			<td>AM9740</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#39; Deadenylase (10 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>DA11101K</td>
			<td></td>
		</tr>
		<tr>
			<td>5X Nucleic acid sample loading buffer</td>
			<td>Bio-Rad</td>
			<td>161-0767</td>
			<td></td>
		</tr>
		<tr>
			<td>8% TBE gel</td>
			<td>Invitrogen</td>
			<td>EC6215BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Invitrogen</td>
			<td>AM9722</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue light transilluminator</td>
			<td>Clare Chemical Research</td>
			<td>DR-46B</td>
			<td></td>
		</tr>
		<tr>
			<td>Chrome-steel beads, 3.2 mm</td>
			<td>BioSpec Products</td>
			<td>11079132c</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogrinder</td>
			<td>Biospec product</td>
			<td>3110BX</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>RPI</td>
			<td>C81040-5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System</td>
			<td>DATAQ Instruments</td>
			<td>DI-245</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) solution mix (10 mM)</td>
			<td>NEB</td>
			<td>N0447L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Invitrogen</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient fractionation system</td>
			<td>Brandel</td>
			<td>BR-184X</td>
			<td></td>
		</tr>
		<tr>
			<td>High-fidelity DNA polymerase (2,000 U/mL)</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td>Supplied with 5X Phusion HF Buffer</td>
		</tr>
		<tr>
			<td>Next-generation sequencing library quantification kit</td>
			<td>Kapa Biosystems</td>
			<td>KK4824</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic acid gel stain</td>
			<td>Invitrogen</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) mRNA isolation kit</td>
			<td>Invitrogen</td>
			<td>61011</td>
			<td></td>
		</tr>
		<tr>
			<td>Rec J exonuclease (10 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>RJ411250</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcriptase (200 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>18080093</td>
			<td>Supplied with 5X first-strand buffer and 0.1 M DTT</td>
		</tr>
		<tr>
			<td>RNA fragmentation buffer</td>
			<td>NEB</td>
			<td>E6186A</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase I (100 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>AM2295</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase inhibitor (20 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>AM2696</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber caps</td>
			<td>BioSpec Products</td>
			<td>2008</td>
			<td></td>
		</tr>
		<tr>
			<td>ssDNA ligase (100 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>CL9021K</td>
			<td>Supplied with 10X CircLigase II buffer and 50 mM MnCl2</td>
		</tr>
		<tr>
			<td>Stainless steel microvials, 1.8 mL</td>
			<td>BioSpec Products</td>
			<td>2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060-5000.0</td>
			<td></td>
		</tr>
		<tr>
			<td>SW-41 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase (10,000 U/mL)</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td>Supplied with 10X T4 polynucleotide kinase buffer</td>
		</tr>
		<tr>
			<td>T4 RNA ligase 2 truncated KQ (200,000 U/mL)</td>
			<td>NEB</td>
			<td>M0373S</td>
			<td>Supplied with 10X T4 RNA ligase buffer and 50% PEG8000</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio-Rad</td>
			<td>1851148</td>
			<td></td>
		</tr>
		<tr>
			<td>Thinwall polyallomer tubes, 13.2 mL</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UV monitor</td>
			<td>Bio-Rad</td>
			<td>7318160</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae strain BY4741</td>
			<td>Open Biosystems</td>
			<td>YSC1048</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide quantification of translation in budding yeast by ribosome profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Detailed pipelines for bioinformatic analysis of ribosome profiling data have been described previously 8,9. In addition, several research groups have developed bioinformatics tools for differential gene expression analysis and processing of sequencing data, which are specific for ribosome profiling method 10,11,12,<sup class="...

Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

The overall goal of this procedure is to identify which regions of mRNA are translated into proteins quantitatively measure protein translation in budding yeast saccharomyces cerevisiae at the genome-wide level. Ribosome profiling can be used to monitor the rate of proteins and disease and to study the mechanisms of translational regulation. The main advantage of this technique is that it adds much greater sensitivity compared to other methods, and of those, measuring proteins and disease in vivo at the single contact resolution.Though this protocol's specific for budding yeast, it is also useful for researchers who are trying to establish ribosome profiling in other model systems, such as mammalian cells and tissues. Extracts will be prepared from yeast cells grown to mid log phase. Use a glass holder filter assembly to filter the culture through 0.45 micromembrane filters.Scrape the pellet with the spatula, flash freeze in liquid nitrogen, and store at minus 80 degrees celsius. Put three chrome steel 3.2 millimeter beads into a 1.8 milliliter stainless steel tube and pre-chill the tube in liquid nitrogen. Add the frozen pellets and cover the steel tube with a silicone rubber cap.Homogenize the sample by cryo-grinding for 10 seconds at 4200 rpm. To keep the samples frozen during the entire grinding procedure it is critical to chill the tube in liquid nitrogen for at least 10 seconds in between each grinding. When the homogenization is done, add one milliliter of lysis buffer that contains cycloheximide to the sample.Mix well by pipetting and transfer the sample to a new 1.5 milliliter tube. Centrifuge at 20, 000 times g at four degrees Celsius for five minutes. Transfer the supernatant into a new 1.5 milliliter tube.Transfer 100 microliters of lysate to another new 1.5 milliliter tube for poly(A)mRNA isolation. Flash freeze the tubes with the mRNA samples on the rest of the lysate, which will be used for footprint extraction in liquid nitrogen, and store at minus 80 degrees celsius. On the night before the footprint extraction, thaw previously prepared sucrose gradients at four degrees celsius.On the following day, thaw the cell lysate on ice. Transfer an aliquot of cell lysate containing 50 OD260 units to a new 1.5 milliliter tube, add fresh lysis buffer up to one milliliter and 10 microliters of RNase one. Incubate at room temperature with gentle rotation on a head over heels rotator for one hour.Next, gently layer the lysate sample on top of a 10%to 50%sucrose gradient in polymer tubes. Using an SW41 ti rotor, ultra centrifuge at 35, 000 rpm at four degrees celsius for three hours. During the ultra cetrifugation, set up the gradient fractionation system.Turn on the components and allow the UV monitor to warm up for at least 30 minutes. Fill the syringe with chase solution and remove any air bubbles inside the syringe and canula. When the ultracentrifugation is complete, install and pierce the ultracentrifuge tube containing the sucrose gradient.Start the syringe pump at one milliliter per minute and collect one milliliter fractions while monitoring the A254 values. Fractions representing the ADS monosome peak are collected and pooled. Filter the fractions through 0.5 milliliter centrifugal filters at 12, 000 times g at four degrees celsius for 10 minutes, discard the flow-through, and repeat until the volume is less than 100 microliters.At 400 microliters of release buffer mixed by pipetting and incubate on ice for 10 minutes, transfer the unit into a new collection tube and centrifuge at 12, 000 times g at four degrees celsius for 10 minutes. Collect the flow-through. Subsequently, purify the footprint fragments and precipitate the samples using ethanol precipitation as described in the text protocol.To begin this procedure, centrifuge the precipitated footprint and fragmented mRNA samples at 20, 000 times g and four degrees celsius for 30 minutes. After removing ethanol and air drying the pellets, we suspend each pellet in 7.75 microliters of water. To each pellet, add one microliter of 10XT4 polynucleotide kinase buffer, one microliter of T4 polynucleotide kinase, and 0.25 microliters of RNase inhibitor.Incubate at 37 degrees celsius for one hour. Add 10 microliters of 2X TBE urea sample buffer to 10 microliters of each footprint and mRNA sample. Incubate the samples and appropriate controls at 75 degrees celsius for three minutes, spin down, and put on ice for one minute.Load each sample into two wells of a 15%TBE urea gel and separate the RNA fragments by gel electrophoresis at 180 volts for one hour. Stain the gel with a nucleic acid gel stain, protected from light for five minutes. While visualizing the gel under a blue light transilluminator, excise the proper band between 28 and 32 nucleotides for footprint samples and around 50 to 70 nucleotides for mRNA samples.Freeze the excised polyacrylamide gel pieces at minus 80 degrees celsius for at least 10 minutes before extracting RNA from the polyacrylamide gel. Prior to reverse transcription, perform three prime adapter ligation and precipitate the samples as described in the text protocol. Resuspend each pellet in 11.5 microliters of nuclease-free water.Then add 0.5 microliters of eight micromolar reverse transcription primer and one microliter of dNTP mix. Incubate at 65 degrees celsius for five minutes and then place on ice. To each sample, add four microliters of 5x first strand buffer, two microliters of 0.1 molar DTT, 0.5 microliters of RNase inhibitor, and 0.5 microliters of reverse transcriptase.Incubate at 48 degrees celsius for 30 minutes, 65 degrees celsius for one minute, and 80 degrees celsius for five minutes. Proceed immediately to the hydrolysis of RNA by adding 0.8 microliters of two molar sodium hydroxide to each sample and incubating at 98 degrees celsius for 30 minutes. Add 0.8 microliters of two molar hydrogen chloride to neutralize the reaction.After precipitating the samples as described in the text protocol, resuspend each pellet in five microliters of nuclease-free water and add five microliters of 2x TBE urea sample buffer. Incubate at 75 degrees celsius for three minutes, spin down, and ice for one minute. Load the sample in one well of a 10%TBE urea gel.Separate the products of the reverse transcription reaction by gel electrophoresis 180 volts for 50 minutes. Stain the gel with a nucleic acid gel stain protected from light for five minutes. Using a blue light transilluminator, excise the band around 128 nucleotides and higher.Freeze the polyacrylamide gel pieces at minus 80 degrees celsius for at least 10 minutes before extracting DNA from the polyacrylamide gel. To circularize the DNA, resuspend each DNA pellet in 16.75 microliters of nuclease-free water. Add two microliters of 10x single-stranded DNA lygase buffer, one microliter of 50 millimolar manganese chloride, and 0.25 microliters of single-stranded DNA Ligase.Incubate at 60 degrees celsius for one hour, followed by 80 degrees celsius for 10 minutes. Without precipitating, store the single-stranded DNA ligation reaction product at minus 20 degrees celsius. Subsequently, perform PCR library amplification followed by library quantification and high throughput sequencing as described in the text protocol.Representative bio analyzer profiles of the PCR amplified footprint library and the sequencing library obtained for mRNA samples are shown. The expected average size of the footprint library is 148 to 152 nucleotides, and the expected size of the mRNA library is 170 to 190 nucleotides. This graph shows the combined number of mRNA, footprint, and rRNA reads obtained for two independent biological replicates of a wild type sample and the hbs1 deletion mutant.50%to 60%of all sequenced reads in the footprint libraries corresponded to ribosome-protected footprint fragments. In contrast, only 3%of rRNA fragments were observed in mRNA libraries, demonstrating that poly(A)mRNA isolation allows effective elimination of rRNA reads. Both footprint and mRNA sample libraries show good reproducibility as indicated by a Pearson correlation coefficient of about 0.99 between matched samples.When combined with mRNA abundance measurements, ribosome profiling allows measuring changes in translation efficiency between experimental conditions. Significantly up-regulated and down-regulated genes in the hbs1 deletion mutant are grouped in accordance to whether they are affected by a change in mRNA transcription, translation efficiency, or by a common effect. Once mastered, the entire protocol can be performed in approximately 11 days.While attempting this procedure, it is important to remember to wear gloves and perform all steps using RNase free reagents and consumables to avoid RNase contamination and lower the risk of RNA degradation in the samples. After watching this video, you should have a good understanding of how to generate ribosome footprints using nuclease digestion, isolate intact ribosome footprint complexes by sucrose gradient fractionation, and prepare DNA libraries for deep sequencing. Don't forget that working with liquid nitrogen can be extremely hazardous, and precautions such as wearing safety glasses and cryo-gloves should always be taken while handling extremely cold materials.

genome-wide-quantification-translation-budding-yeast-ribosome

Research

JoVE Journal

Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.