We thank the financial support from Genome Canada and Genome British Columbia for operations (204PRO) and technology development (214PRO) through the Genome Innovations Network (GIN). We thank the Drug Discovery Platform at the Research Institute of the McGill University Health Center for the use of the MALDI-TOF instrument for filming. H.L. is grateful for support from a postdoctoral fellowship from the National Science and Engineering Research Council of Canada (NSERC). C.H.B is grateful for support from the Leading Edge Endowment Fund (LEEF). C.H.B. is grateful for support from the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, Quebec, Canada). M.X.C. and C.H.B. are grateful for support from the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, Quebec, Canada).

The amounts of the reagents described below are based on the measurement of 20 patient plasma samples. The protocol presented below follows the guidelines of the University of Victoria&#39;s human research ethics committee.
1. Generation of Ang I in Human Plasma
<ol>
	<li>Thaw plasma samples (&#8805;200 &#181;L) in a room temperature water bath for 5 min, and then put the samples on ice until completely thawed.</li>
	<li>Transfer 200 &#181;L of each plasma sample manually to separate wells o...

<ol>
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Proteomics. 107, 24-30 (2014).</li><li>Landegren, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+for+Sensitive+Plasma+Proteome+Analysis.">Opportunities for Sensitive Plasma Proteome Analysis.</a> Anal. Chem. 84 (4), 1824-1830 (2012).</li><li>Guo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoaffinity+Enrichment+and+Mass+Spectrometry+Analysis+of+Protein+Methylation.">Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation.</a> Mol. &amp; Cell. Proteomics. 13 (1), 372-387 (2014).</li><li>Florentinus-Mefailoski, A., Soosaipillai, A., Dufresne, J., Diamandis, E. P., Marshall, J. 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Compared to conventional MS-based protein quantification, iMALDI uses antibodies to enrich the analytes and purify them from complex samples, therefore making it possible to quantify proteins or peptides at low concentrations. A critical step in the iMALDI protocol is the immuno-enrichment of the target peptides. For this purpose, antibodies with high specificity and affinity should be selected. In SISCAPA, it has been reported that antibody affinities at 10-9 M or better would be needed to achieve high sensit...

Mass spectrometry has become an indispensable tool in quantitative proteomics. Mass spectrometry can determine the masses of target proteins or peptides, therefore the obtained analyte signals can be highly specific compared to immunoassays. Two ionization methods, electrospray and MALDI, are most commonly used for detecting proteins and peptides1,2,3,4. A major challenge in MS-based protein quantification lies in the detection of low-abundance proteins in complex samples at ng/mL or pg/mL concentrations in the presence of high-abundance proteins, and many candidate protein biomarkers found in human plasma are within this range5. This problem is largely caused by the inherently wide dynamic range and complexity of the human proteome6.
To overcome these detection challenges, immuno-MS methods have been developed to enrich the target proteins or peptides from the sample solutions onto a solid surface, followed by elution of the analytes and MS measurement7,8,9,10. Through immuno-enrichment, the analytes are purified from complex samples and therefore the ion-suppression effects from other molecules are minimized. Among various solid supports, magnetic beads are currently most widely used as they have the advantages of high antibody binding capacity and ease of handling. Magnetic beads with different functionalizations and sizes have been developed and commercialized for immunoprecipitation experiments. To date, immuno-enrichment on beads has been interfaced with both electrospray ionization (ESI) and MALDI-MS for protein and peptide measurement. In stable isotope standards and capture by anti-peptide antibodies (SISCAPA) technology, proteins in the samples are digested, followed by incubation with antibody-coated beads for immuno-enrichment. In &#34;classical&#34; SISCAPA, the captured proteotypic peptides are eluted from the beads, and measured by Liquid Chromatography-ESI-MS (LC-MS), or by direct infusion ESI-Multiple Reaction Monitoring-MS (ESI-MRM-MS)11,12. Immuno-enrichment improved the MRM assay sensitivity by 3-4 orders of magnitude, reaching the low ng/mL range13.
Compared to electrospray-MS, MALDI-MS is faster, and does not involve the cleaning and re-equilibration of LC columns so there are no carryover and contamination issues, making it more suitable for high-throughput studies14. Immuno-MALDI technology has been developed in our laboratory to combine immuno-enrichment with MALDI-MS for sensitive and specific quantification of peptides and proteins (based on quantitation of proteotypic peptides)15,16,17. After immuno-enrichment, the beads are deposited on a MALDI target plate, the matrix solution is added to beads, and the plate is ready for analysis by a MALDI-TOF-MS after drying. Elution of the peptides from the beads is not performed as a separate step, but affinity-bound analytes are eluted by the MALDI matrix solution when it is added to the bead spots, thereby simplifying the sample preparation and minimizing sample loss. The iMALDI technology has been applied in a variety of applications18,19, and recently has been automated and used for measuring Angiotensin I (Ang I) for determining plasma renin activity (PRA)20. This protocol will demonstrate the procedure used to perform an automated iMALDI assay. Taking the PRA assay as an example, inter-day CVs of less than 10% have been achieved through automation, with the capability of measuring 744 samples per day20.
The iMALDI PRA assay demonstrated in this manuscript does not require protein digestion, as the target molecule (Ang I) is a peptide with a molecular weight of 1295.7 Da. In other assays where protein digestion is necessary and a peptide is used as the surrogate for the intact protein, the selected peptide for iMALDI should be unique and specific to the target protein, with a mass over 800 Da so that it can be easily distinguished from the chemical noise from the MALDI matrix solution. Anti-peptide antibodies are required for the immuno-enrichment of the peptides. The protocol for an iMALDI assay measuring PRA consists of four steps: 1) generation of Ang I in human plasma; 2) immuno-enrichment of Ang I using antibody-coated beads; 3) transfer of beads to a MALDI target plate and adding matrix solution; and 4) signal acquisition by a MALDI-TOF-MS and data analysis20.

<table width="892">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Healthy control human plasma</td>
			<td class="xl63" style="width:134pt" width="178">Bioreclamation</td>
			<td class="xl68" style="width:89pt" width="119">HMPLEDTA2</td>
			<td class="xl66" style="width:242pt" width="323"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ammonium bicarbonate</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl69">09830</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ammonium citrate dibasic</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">09833</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">CHAPS (&#62;=98%)</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">C9426</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Albumin from chicken egg white (&#62;98%)</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">A5503</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ethylenediaminetetraacetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">EDS</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Alpha-cyano-4-hydroxycinnamic acid</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">70990</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Phosphate buffered saline</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">P4417</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Phenylmethanesulfonyl fluoride</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">78830</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Trifluoroacetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">85172</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">acetonitrile</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">34967</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">water</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">39253</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">acetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">320099</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Tris(hydroxymethyl)aminomethane</td>
			<td class="xl63" style="width:134pt" width="178">Roche Diagnostics</td>
			<td class="xl71">3118169001</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Dynabeads Protein G magnetic beads</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">10003D</td>
			<td class="xl66">2.8 &#956;m, 30 mg/mL</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">anti-Ang I goat polyclonal antibody</td>
			<td class="xl63" style="width:134pt" width="178">Santa Cruz Biotechnology</td>
			<td class="xl68" style="width:89pt" width="119">sc-7419</td>
			<td class="xl66"></td>
		</tr>
		<tr height="84" style="height:63.0pt">
			<td class="xl63" height="84" style="width:204pt;height:63.0pt" width="272">Nat and SIS Ang I</td>
			<td class="xl63" style="width:134pt" width="178">synthesized at the University of Victoria-Genome BC Proteomics Centre</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Automated liquid handling system</td>
			<td class="xl63" style="width:134pt" width="178">Agilent</td>
			<td class="xl68" style="width:89pt" width="119">16050-102</td>
			<td class="xl66">Agilent Bravo robotic workstation</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">12321D</td>
			<td class="xl66">Invitrogen DynaMag-2 magnet</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Tube rotator</td>
			<td class="xl63" style="width:134pt" width="178">Theromo Scientific</td>
			<td class="xl68" style="width:89pt" width="119">400110Q</td>
			<td class="xl66">Labquake Tube Rotator</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">12027</td>
			<td class="xl66">DynaMag-96 side skirted magnet</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">VP Scientific</td>
			<td class="xl68" style="width:89pt" width="119">771RM-1</td>
			<td class="xl66">used to pull the beads to the bottom of the well</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">MALDI-TOF</td>
			<td class="xl63" style="width:134pt" width="178">Bruker</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl66">Bruker Microflex LRF instrument</td>
		</tr>
	</tbody>
</table>

peptide and protein quantification using automated immuno-maldi (imaldi)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

An automated iMALDI procedure for measuring Ang I is shown in Figure 1. Target peptides (either endogenous peptides or peptides from digested proteins) are enriched on anti-peptide magnetic beads, and then the beads are transferred to a target plate for MALDI measurement. The whole procedure is simplified compared to other immuno-MS technologies that require additional peptide elution steps. Automation of the iMALDI assay allows for high-throughput analysis o...

Watch this Scientific Journal Video about Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI at JoVE.com

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

The overall goal of this automated immuno-MALDI assay is to quantify peptides and intact proteins in complex biological fluids in an accurate and sensitive manner. This method can help answer key questions in the quantitative proteomics field, such as quantify protein levels for diagnostics or for monitoring a patient's response to treatment. The key advantage of this method is that it can quantify protein levels very sensitively and accurately from complex samples.This technique allows the diagnosis of diseases at an early stage because the immuno-enrichment step of the assay allows quantitation of low abundant protein and peptide biomarkers. Though this method can provide insight into plasma renin activity, it can also be applied to other disease-related studies, such as quantification of cancer-related proteins for prognosis, diagnosis, and treatment management. To begin this procedure, thaw human plasma samples in a room temperature water bath for five minutes.Then, place the samples on ice until completely thawed. Next, manually transfer 200 microliters of each plasma sample to separate wells of a 1.1 milliliter deep well plate. Centrifuge the plate for 10 minutes at two degrees Celsius and 3, 000 rpm.Then, use the Bravo to serially dilute a 500 femtomole per microliter Ang-1 NAT standard solution to prepare six calibrator solutions with chicken egg white albumin. Following centrifugation, pipette 200 microliters of each calibrator to a well and 125 microliters of generation buffer to the sample plate. Using an automated liquid handling system, mix 125 microliters of plasma supernatant or 125 microliters of the chicken egg white albumin solution with 25 microliters of angiotensin I generation buffer in a new 1.1 milliliter deep well plate.Now, automatically transfer the solutions to a 96-well plate with three replicates per solution and 34 microliters of solution per well. Then, incubate the plate at 37 degrees Celsius for three hours. For conjugation of the antibody with protein G beads, first transfer 78 microliters of previously prepared bead slurry to a 1.5 milliliter tube.Wash the beads seven times with one milliliter of 25%acetonitrile in PBSC and three times with one milliliter of PBSC, using a magnetic stand to pellet the beads between each washing. After washing the beads, resuspend them in 110 microliters of PBSC and add 110 microliters of anti-angiotensin I antibody. Mix the beads and solution by pipetting.Then, incubate the beads at room temperature for one hour while rotating at eight rpm. Following this, wash the beads three times with one milliliter of PBSC. Then, resuspend the beads in 1, 100 microliters of PBSC.Now, manually transfer the bead solution to a 96-well plate. Then, have the Bravo aliquot the beads to the same 96-well plate. After the three-hour angiotensin I generation period, place the incubation plate on ice for 10 minutes to terminate the generation of angiotensin I.Automatically dilute a stable isotope standards peptide stock solution 100-fold with PBS buffer.Further, automatically aliquot the stable isotope standard peptide dilution to a 96-well PCR plate. Transfer 10 microliters of the diluted peptide solution to each well of the incubation plate. Mix the diluted peptide solution with the plasma samples or the chicken egg white albumin solution.Automatically add the sample or standard solutions to the bead solutions to the incubation plate and mix. After incubating one hour, automatically wash the beads three times with five millimolar ammonium bicarbonate solution. Use a magnet to pull the beads to the bottom after each wash.After the last wash, add 10 microliters of the ammonium bicarbonate solution to each well to resuspend the beads. Automatically transfer seven microliters of the bead slurry onto a MALDI target plate with a spot size of 2, 600 micrometers. Once the beads have dried, automatically add two microliters of previously prepared HCCA matrix solution from the matrix well onto each sample spot on the target plate.Following this, analyze the sample spots with a MALDI-TOF instrument using positive reflector mode. Perform internal calibration, data smoothing, and baseline subtraction with the vendor-specific software. The automated iMALDI procedure for measuring angiotensin I allows for high-throughput analysis of a large number of samples with an intraday coefficient of variation below 10%Representative spectra obtained by measuring angiotensin I in human plasma samples are displayed here.Correlation of PRA values from 188 patient samples obtained with the automated iMALDI assay with PRA values obtained using a clinical LC-MS/MS procedure is shown here. The two methods have a correlation coefficient of 0.98, and the difference between the slopes may be due to the use of different internal standards or different antibodies. The linear range of the assay and the assay precision are displayed here.Once mastered, this technique can be performed in approximately five hours. While attempting this procedure, remember to properly adjust the tip head and the liquid handling speed. After this procedure, additional methods can be used to confirm primary aldosteronism in patients, including oral salt loading or saline infusion tests.The automated iMALDI technique allows the specific and accurate high-throughout quantitative screening of biological samples, including patient samples, for disease-related proteins or peptides. After watching this video, you should have a good understanding of how to perform an automate iMALDI assay for mass spectrometry-based protein and peptide quantification. Don't forget that working with biological samples, such as human plasma, is extremely hazardous, and the necessary safety precautions must always be taken while performing the procedure.

peptide-and-protein-quantification-using-automated-immuno-maldi-imaldi

Research

JoVE Journal

Chemistry

7.9K ビュー.  University of Victoria-Genome BC Proteomics Centre. The overall goal of this automated immuno-MALDI assay is to quantify peptides and intact proteins in complex biological fluids in an accurate and sensitive manner. This method can help answer key questions in the quantitative proteomics field, such as quantify protein levels for diagnostics or for monitoring a patient's response to treatment. The key advantage of this method is that it can quantify protein levels very sensitively and accurately from complex samples.This technique allow...

ビデオ: Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

identifying protein-protein interaction sites using peptide arrays

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide...

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated...

using the open-source maldi tof-ms idbac pipeline for analysis of microbial protein and specialized metabolite data

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite...

quantitation of protein expression and co-localization using multiplexed immuno-histochemical staining and multispectral imaging

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral...

specificity analysis of protein lysine methyltransferases using spot peptide arrays

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the...

automated quantification and analysis of cell counting procedures using imagej plugins

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter.  Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the...

protein kinase c-delta inhibitor peptide formulation using gold nanoparticles

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated...

automated image-based quantification of neutrophil extracellular traps using netquant

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence...

automated multiplex immunofluorescence panel for immuno-oncology studies on formalin-fixed carcinoma tissue specimens

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology...

detection of protein ubiquitination sites by peptide enrichment and mass spectrometry

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome...

proofreading and dna repair assay using single nucleotide extension and maldi-tof mass spectrometry analysis

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

quantification of site-specific protein lysine acetylation and succinylation stoichiometry using data-independent acquisition mass spectrometry

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are...

detection and quantification of calcitonin gene-related peptide (cgrp) in human plasma using a modified enzyme-linked immunosorbent assay

Detection and Quantification of Calcitonin Gene-Related Peptide CGRP in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human...

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

automated imaging and analysis for the quantification of fluorescently labeled macropinosomes

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate...

biotinylated cell-penetrating peptide probe to detect protein-protein interactions

This video demonstrates a technique that uses biotinylated cell-penetrating peptides to detect intracellular protein-protein interactions. The cell-penetrating peptides facilitate the conjugated peptide to enter a cell and specifically bind to its interacting proteins. The biotin, after binding to avidin, allows purification of the interacting proteins&#8217; complex from a cell...

Biotinylated Cell-Penetrating Peptide Probe to Detect Protein-Protein Interactions

sample preparation for metabolic profiling using maldi mass spectrometry imaging

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

semi-automated planimetric quantification of dental plaque using an intraoral fluorescence camera

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research...

automated quantification of synaptic fluorescence in c. elegans

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane...

Automated Quantification of Synaptic Fluorescence in C. elegans

visualizing subcellular localization of a protein in the heart using quantum dots-mediated immuno-labeling followed by transmission electron microscopy

Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy

The present protocol describes a method of immuno-labeling a protein in the heart tissue sections using quantum dots. This technique provides a useful tool to visualize any protein&#39;s subcellular localization and expression at the ultrastructural...

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...