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 of a 1.1 mL deep-well plate (sample plate), and centrifuge the plate in a centrifuge for 10 min at 2 &#176;C and 1278 x g.</li>
	<li>Use an automated liquid handling system to serially dilute a 500 fmol/&#181;L Ang I NAT standard solution to prepare six calibrator solutions with chicken egg white albumin (0.1, 0.2, 0.6, 1.9, 5.7, 17.2 fmol/&#181;L).</li>
	<li>Pipette 200 &#181;L of each calibrator to a well, and 125 &#181;L of generation buffer to sample plate. 
	NOTE: The CEWA in phosphate buffered saline (PBS) buffer needs to be freshly prepared on the day of the experiment.</li>
	<li>Using an automated liquid handling system, in a new plate mix 125 &#181;L of plasma supernatant or 125 &#181;L of CEWA in PBS with 25 &#181;L of Ang I generation buffer, which contains 1 M Tris, 0.2 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM phenylmethylsulfonyl fluoride (PMSF). 
	NOTE: Prepare the Ang I generation buffer by mixing a 1 M Tris/0.2 mM EDTA aqueous buffer (adjust to pH 5.5 using acetic acid) and a PMSF solution (100 mM in methanol). Both solutions can be stored up to 1 month at 4 &#176;C. Mix the two solutions on the day of the experiment.</li>
	<li>Automatically transfer the solutions into a 96-well plate, 3 replicates per solution, 34 &#181;L per well. Incubate the plate at 37 &#176;C for 3 h.</li>
</ol>
2. Immuno-enrichment of Ang I Using Antibody-coated Beads
<ol>
	<li>Conjugation of antibody onto magnetic Protein G beads 
	NOTE: Conjugation of the antibody with the beads is performed manually during the 3 h Ang I generation period on the day of the experiment. For other analytes, the conjugated beads might be able to be stored in PBS buffer containing 0.015% CHAPS (PBSC) at 4 &#176;C for three months or longer, depending on the properties and stability of the antibody.
	<ol>
		<li>Transfer 110 &#181;L of bead slurry (enough for measuring 20 samples and making a 6-point standard curve) into a 1.5 mL tube. Wash the beads seven times with 1 mL of 25% acetonitrile/PBSC, and three times with 1 mL of PBSC. Use a magnetic stand to pellet the beads between each washing step. Remove the wash buffer after the last wash. 
		NOTE: Washing 7 times with 25% acetonitrile/PBSC is critical for removing the MS-incompatible additives in the bead slurry, such as Tween-20. If there are no such additives in the selected beads, this extensive washing step might not be necessary.</li>
		<li>Resuspend the beads in 110 &#181;L of PBSC, and add 110 &#181;L of anti-Ang I antibody (final antibody concentration: 100 &#181;g/mL). Mix the beads and solution by pipetting, and then incubate them at room temperature for 1 h, rotating at 8 rpm.</li>
		<li>Wash the beads 3 times with 1 mL of PBSC, and resuspend in 1100 &#181;L of PBSC. 
		NOTE: If any bead solution has flowed into the cap of the tube during incubation, spin down the solution using a benchtop centrifuge at 2680 x g.</li>
		<li>Transfer the bead solution manually to a 96-well plate (bead standard plate). Use the automated liquid handling system to aliquot the beads to the same 96-well plate, 120 &#181;L per well.&#160;</li>
	</ol>
	</li>
	<li>Immuno-enrichment on the beads
	<ol>
		<li>After the 3 h Ang I generation period, place the incubation plate on ice for 10 min to terminate the generation of Ang I.</li>
		<li>Automatically dilute the SIS peptide stock solution (10 pmol/&#956;L) 100-fold with PBS buffer, and further automatically aliquot the stable isoptope standard peptide dilution to a 96-well PCR plate. Transfer 1.5 &#181;L of a stable isotope standards (SIS) peptide solution (containing 100 fmol) into each well of the incubation plate and mix it with the plasma samples or the CEWA in PBS buffers.</li>
		<li>Automatically transfer the contents of the incubation plate to the bead solution, 10 mL per well, mix.</li>
		<li>Incubate the plate at 4 &#176;C for 1 h while rotating at 8 rpm.</li>
		<li>Wash the beads three times automatically with 5 mM ammonium bicarbonate (AmBic) solution, 100 &#181;L per well per wash. After the last wash, resuspend the beads in 7 &#181;L of 5 mM AmBic solution in each well. Use a magnet to pull the beads to the bottom after the last wash.</li>
	</ol>
	</li>
</ol>
3. Transfer of Beads onto a MALDI Target Plate and Adding Matrix Solution
<ol>
	<li>Transfer 7 &#181;L of the bead slurry automatically onto a MALDI target plate with a spot size of 2,600 &#181;m. Let the beads dry out. 
	NOTE: A small USB-powered fan can be used to accelerate the bead drying process.</li>
	<li>Automatically add 2 &#181;L of &#945;-cyano-4-hydroxycinnamic acid (HCCA)-matrix solution (containing 3 mg/mL HCCA, 1.8 mg/mL ammonium citrate, 70% acetonitrile, and 0.1% trifluoroacetic acid) from the matrix well onto each sample spot on the target plate.</li>
</ol>
4. Signal Acquisition by a MALDI-TOF-MS and Data Analysis
<ol>
	<li>Analyze the sample spots with a MALDI-TOF instrument using positive reflector mode. Perform internal calibration, data smoothing, and baseline subtraction automatically with vendor-specific software. 
	NOTE: The mode (positive/negative, linear/reflector) selected for MALDI-TOF measurement depends on the target peptides or proteins.</li>
	<li>Calculate the relative response ratio (Nat/SIS intensity ratio) and compare it to the standard curve to determine of the Ang I concentration in each sample. Calculate PRA using Equation (1), where &#916;tAng I generation represents the time used for the generation of Ang I. 
	PRA = [Ang I]/&#916;tAng I generation (1)</li>
</ol>

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C.H.B holds the patent on the iMALDI technology.

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 sensitivity at low ng/mL21. In addition, beads with high antibody binding capacity are preferred for optimal enrichment.
It is also important to generate relatively &#34;clean&#34; spectra for the target molecules with minimal background levels. High signal-to-noise ratios can be achieved through extensive bead washing after the immuno-enrichment step. Also, we have found that washing the bead spots on the target plate after adding and drying the matrix solution can significantly reduce the noise signals and thus improve the signal-to-noise ratios.
The iMALDI PRA assay demonstrated in this work does not require the proteolysis steps, as the target molecule Ang I is a peptide. To quantify protein molecules in a sample, the protein is typically digested into peptides before immuno-enrichment, although for epitope-containing peptides, the digestion can be performed on the captured protein22,23. In this case, an existing protein digestion protocol can be easily inserted into the iMALDI workflow.&#160; Depending on the digestion buffer, a desalting step might be needed before capture of the peptides on beads, to avoid interference with the antibody-peptide binding.
Similar to other immuno-MS methods, production of anti-peptide antibodies is a major challenge in the development of iMALDI assays. Recently, Triple X Proteomics technology has been developed to produce affinity binders that can target multiple peptides per binder24. This may reduce the cost of anti-peptide antibodies, and would facilitate the development of simultaneous multiplexed assays using a single incubation. Also, antibody fragments25 or synthetic aptamers26 could be alternatives to the use of intact antibodies and would have the advantage of easier production. Synthetic aptamers have been reported as showing lower background levels than intact antibodies when used for affinity-enrichment26.
iMALDI technology combines the advantages of immunoassays with MALDI mass spectrometry, enabling rapid, high-throughput protein/peptide analysis in a large number of complex samples, with high sensitivity and specificity. Unlike other immuno-MS methods, after immuno-capture, the target peptides are not eluted in the tube, but the beads carrying analytes are transferred onto the MALDI target plate for MS-analysis, thus avoiding the loss of peptides due to their adsorption to plastic tubes. Compared to widely used proteomic tools such as western blot or ELISA, only one antibody is required in iMALDI, significantly reducing the assay development time and cost. The lower limit of detection we have obtained in the PRA assay is comparable to conventional ELISA, and well within the physiological range. The antibody and the mass-to-charge ratio provide a double selection process, which ensures the high specificity of the assay. Applications of iMALDI is not limited to PRA assays, but can be applied for quantitation of any protein expression and even quantification of protein modification in complex samples. Notably, the automated high-throughput iMALDI could be a powerful clinical tool for the discovery and validation of protein biomarkers in a variety of diseases, due to the presence of benchtop MALDI instruments currently used in clinics for bacterial identification.

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><tbody><tr><td>Healthy control human plasma</td><td>Bioreclamation</td><td>HMPLEDTA2</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma Aldrich</td><td>09830</td><td></td></tr><tr><td>Ammonium citrate dibasic</td><td>Sigma Aldrich</td><td>09833</td><td></td></tr><tr><td>CHAPS (&gt;=98%)</td><td>Sigma Aldrich</td><td>C9426</td><td></td></tr><tr><td>Albumin from chicken egg white (&gt;98%)</td><td>Sigma Aldrich</td><td>A5503</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma Aldrich</td><td>EDS</td><td></td></tr><tr><td>Alpha-cyano-4-hydroxycinnamic acid</td><td>Sigma Aldrich</td><td>70990</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma Aldrich</td><td>78830</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Thermo Fisher Scientific</td><td>85172</td><td>LC-MS grade</td></tr><tr><td>acetonitrile</td><td>Fluka</td><td>34967</td><td>LC-MS grade</td></tr><tr><td>water</td><td>Fluka</td><td>39253</td><td>LC-MS grade</td></tr><tr><td>acetic acid</td><td>Fluka</td><td>320099</td><td>LC-MS grade</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Roche Diagnostics</td><td>3118169001</td><td></td></tr><tr><td>Dynabeads Protein G magnetic beads</td><td>Thermo Fisher Scientific</td><td>10003D</td><td>2.8 &lt;span class=&quot;font5&quot;&gt;&amp;mu;m, 30 mg/mL&lt;/span&gt;</td></tr><tr><td>anti-Ang I goat polyclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-7419</td><td></td></tr><tr><td>Nat and SIS Ang I</td><td>synthesized at the University of Victoria-Genome BC Proteomics Centre</td><td></td><td></td></tr><tr><td>Automated liquid handling system</td><td>Agilent</td><td>16050-102</td><td>Agilent Bravo robotic workstation</td></tr><tr><td>Magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td>Invitrogen DynaMag-2 magnet</td></tr><tr><td>Tube rotator</td><td>Theromo Scientific</td><td>400110Q</td><td>Labquake Tube Rotator</td></tr><tr><td>Magnet</td><td>Thermo Fisher Scientific</td><td>12027</td><td>DynaMag-96 side skirted magnet</td></tr><tr><td>Magnet</td><td>VP Scientific</td><td>771RM-1</td><td>used to pull the beads to the bottom of the well</td></tr><tr><td>MALDI-TOF</td><td>Bruker</td><td></td><td>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 of a large number of samples with an inter-day coefficient of variation (CV) below 10% 20. Representative spectra obtained by measuring Ang I in human plasma samples are shown in Figure 2. The SIS peptides function as an internal standard for peptide quantitation. Correlation of PRA values from 188 patient samples obtained with the automated iMALDI assay with PRA values obtained by using a clinical LC-MS/MS procedure17 is shown in Figure 3. The two methods have a correlation coefficient of 0.98; the difference between the slopes might be caused by the use of different internal standards in the two methods, or by the use of different antibodies in iMALDI procedure20. The linear range of the assay and the assay precision are shown in Figure 4 and Figure 5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55933/55933fig1.jpg" /> 
Figure 1: Process flow schematic of an automated iMALDI PRA assay. Adapted from reference20, with permission. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55933/55933fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55933/55933fig2.jpg" /> 
Figure 2: Representative spectra of the NAT and SIS Ang I peptides measured from a human plasma sample.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55933/55933fig3.jpg" /> 
Figure 3: Correlation of PRA values measured by iMALDI and by LC-MS/MS from 188 patient samples. Adapted from reference20, with permission.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55933/55933fig4.jpg" /> 
Figure 4: Linear ranges of the iMALDI PRA assay in both (A) reflector and (B) linear mode. Adapted from reference20, with permission. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55933/55933fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55933/55933fig5.jpg" /> 
Figure 5: Intraday precision (A) and interday precision (B) of iMALDI PRA assays on plasma pools with low, medium, and high PRA values. Adapted from reference20, with permission. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55933/55933fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.9K Views.  University of Victoria-Genome BC Proteomics Centre. A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented. 

Video: Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Bioengineering

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Medicine

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

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 analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Biochemistry

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Immunology and Infection

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome ("surfaceome") in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

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 format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biology

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...