a stage tip procedure for desalting and purification of peptides

A STAGE Tip Procedure for Desalting and Purification of Peptides

To begin, pack three layers of C18 resin in a 200-microliter pipette tip to construct a STop And Go Extraction or STAGE tip for each peptide sample.
Stop the enzymatic digestion of the previously incubated peptide samples by adding 1 by 10 volumes of stopping solution. Centrifuge the samples at 13,200 rpm for 10 minutes and transfer the supernatant to a new 2-milliliter tube.
Next, wash the STAGE tips with 100 microliters of 100 percent acetonitrile and centrifuge the STAGE tips at 1000 g for 2 minutes or until all the liquid pass through the C18 resin.
Repeat the STAGE tip washes with 50 microliters of Buffer B followed by 200 microliters of Buffer A with the same centrifugation conditions.
Load the samples into the STAGE tips to centrifuge at 1000 g for 3 to 5 minutes. Then, wash the STAGE tips with 200 microliters of Buffer A by centrifugation at 1000 g for 3 to 5 minutes.
Next, add 50 microliters of Buffer B into each STAGE tip, and using a syringe, elute the samples containing Buffer B into a 0.2-milliliter PCR tube.
Once the samples are eluted, dry the peptides using a vacuum centrifuge for 45 minutes. Run the samples on high-resolution mass spectrometry with the conditions described in the text manuscript.
To process the data for proteomic profiling, upload the raw data files obtained from mass spectrometry into the MaxQuant software. Refer to the text manuscript for all parameters of the software and its adjustments.
In global parameters, import the FASTA file of Homo sapiens for sequence identification downloaded from the Uniprot database recording the Organism ID, number of proteins, and file download date.
Set the number of computer cores for processing and select Start. Upon completion of MaxQuant, a &#39;Combined&#39; folder, along with other files needed for uploading to data repositories, is generated.
To analyze the data, upload the &#39;proteingroups.txt&#39; into Perseus and select all label-free quantification or LFQ intensity files to the &#39;Main&#39; window. Filter the rows by removing contaminants, reverse peptides, and peptides only identified by site and transform the data by log2(x).
To observe the number of proteins detected in each replicate, construct a numeric Venn diagram and set categorial annotations for each sample by providing the same name for all replicates of one biological sample.
Filter the rows based on valid value with the protein identified in more than 50 percent of the replicates and within at least one group. To replace missing values for LFQ, perform imputation from the normal distribution.
Add annotation information downloaded from the Perseus website to the protein rows by adding txt.gz FASTA files in &#39;bin&#39;, &#39;conf&#39;, and &#39;annotation&#39; folders. Bring in specific terms like protein names, gene names, and gene ontology.
The matrix is now complete and can be visualized in tools such as principal component analysis plots, heat maps, and category enrichment. Perform statistical testing to determine significant changes in protein abundance between the tested conditions.

a-stage-tip-procedure-for-desalting-and-purification-of-peptides

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1. Purification and Desalting of Peptides
<ol>
	<li>Pack three layers of C18 resin in a 200 &#956;l pipette tip for each peptide sample to construct a STop And Go Extraction (STAGE)-tip.</li>
	<li>Add 1/10 stopping solution (20% acetonitrile [ACN)] and 6% trifluoroacetic acid [TFA]) to samples.</li>
	<li>Centrifuge samples for pellet precipitation at 13,200 rpm for 10 min.</li>
	<li>Transfer the supernatant to a new 2 mL tube and discard the pellet.</li>
	<li>Wash stage tips with 100 &#956;l of 100% ACN.</li>
	<li>Centrifuge STAGE tips at 1000 x g for 2 min. 
	NOTE: Centrifuge STAGE tips until all liquid has run through the C18 resin.</li>
	<li>Wash STAGE tips with 50 &#956;l Buffer B (80% ACN, 0.5% Acetic acid, and ddH2O).</li>
	<li>Centrifuge STAGE tips at 1000 x g for 2 min.</li>
	<li>Wash STAGE tips with 200 &#956;l Buffer A (2% ACN, 0.1% TFA, 0.5% Acetic acid, and ddH2O).</li>
	<li>Centrifuge stage tips at 1000 x g for 2 min.</li>
	<li>Load samples into STAGE tips.</li>
	<li>Centrifuge samples at 1000 x g for 3-5 min.</li>
	<li>Wash STAGE tips with 200 &#956;l Buffer A.</li>
	<li>Centrifuge at 1000 x g for 3-5 min.</li>
	<li>Add 50 &#956;l of Buffer B into each STAGE tip</li>
	<li>Using a syringe, elute samples containing Buffer B into a 0.2 mL PCR strip tube.</li>
	<li>Once samples are eluted, dry the peptides using a vacuum centrifuge for 45 mins.</li>
	<li>Once samples are dry, store them at room temperature until MS. 
	NOTE: If peptide storage is needed for more than two weeks, we suggest storing at -20&#176;C.</li>
	<li>Run the sample on a high-resolution mass spectrometry (MS). 
	NOTE: Sample measurement parameters (e.g., amount of sample loaded, liquid chromatography gradient length, resolution) will depend on the instrumentation used.</li>
</ol>
2. Proteomic Profiling of Neutrophils&#160; 
<ol>
	<li>Raw data processing 
	<ol>
		<li>Upload Raw data files obtained from MS into MaxQuant.</li>
		<li>The software is designed to run with bottom-up, data-dependent acquisition files, and many default parameters are optimized for such samples. Default parameters include Digestion (Trypsin P), Variable modifications of methionine oxidation (oxidation (M)) and N-acetylation of proteins (Acetyl (Protein N-term)), and fixed modifications of carbamidomethylation of cysteine (carbamidomethyl (C)).</li>
		<li>Adjustments to these parameters include label-free quantification (LFQ; set minimum ratio count to 1), Match Between Runs (i.e., on/off), and a minimum number of peptides identified per protein (recommend setting to 2).</li>
		<li>In global parameters, import the FASTA file for sequence identification. For this experiment, the Homo sapiens (Human) (UP000005640) FASTA file from the Uniprot database was used. NOTE: Record the following information for record-keeping: Organism ID = 9606; the number of proteins = 79,740; file download date.</li>
		<li>Set the number of computer cores to be used for processing and select Start.</li>
		<li>Upon completion of MaxQuant, a &#39;combined&#39; folder, along with other files, will be generated. 
		NOTE: The other files may be used to identify peptides, modifications, etc., and may be needed to upload the full dataset to data repositories, including Proteome Xchange (PRIDE).</li>
	</ol>
	</li>
	<li>Data analysis and visualization in Perseus
	<ol>
		<li>Upload the &#39;proteingroups.txt&#39; into Perseus and select all LFQ intensity files for the &#39;Main&#39; window. 
		NOTE: Baseline information is imported directly into the Perseus windows, but other information (e.g., modification sites) may need to be added manually.</li>
		<li>Filter rows by removing contaminants, reverse peptides, and peptides only identified by site.</li>
		<li>Transform the data by log2(x).</li>
		<li>To observe the number of proteins detected in each replicate, construct a numeric Venn diagram.</li>
		<li>Set categorial annotations for each sample. 
		NOTE: For statistical purposes, all replicates of one biological sample should have the same name.</li>
		<li>Filter rows based on valid values, with the protein identified in &#62;50% of the replicates and within at least one group. 
		NOTE: Filtering by valid values can be more stringent, depending on the experiment setup and goals (e.g., protein must be present in all samples).</li>
		<li>To replace missing values for LFQ, imputation from the normal distribution can be performed. 
		&#8203;NOTE: the default parameters are optimized for LFQ data but can be adjusted as desired.</li>
		<li>Add annotation information to the protein rows. Add the txt.gz FASTA file (obtained from the Perseus website) in the &#39;bin&#39;, &#39;conf&#39;, and &#39;annotation&#39; folders and bring in specific terms (e.g., protein names, gene names, gene ontology).</li>
		<li>The matrix is now complete and can be used for statistical testing (e.g., ANOVA, t-tests) to determine significant changes in protein abundance between the tested conditions. Other forms of visualization tools include principal component analysis plots, heat maps, and category enrichment. 
		NOTE: Annotation information can also be downloaded from UniProt and integrated into the Perseus framework.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml Falcon Tube&#160;</td>
			<td>VWR</td>
			<td>470225-000</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>14955235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate (ABC)</td>
			<td>ThermoFisher Scientific</td>
			<td>A643-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>1099510001</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td>34850-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile (ACN), MS grade</td>
			<td>Pierce</td>
			<td>TS-51101</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA assay kit&#160;</td>
			<td>VWR</td>
			<td>CA62408-940</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 disks (STAGE tips)</td>
			<td>Fisher Scientific&#160;</td>
			<td>13110019</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Sigma Aldrich</td>
			<td>EP022628188</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>ThermoFisher Scientific</td>
			<td>R0861</td>
			<td>Prepare 1M stock of DTT aliquots and store at&#160; -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>12483020</td>
			<td>Thaw out, prepare aliquots and stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Prepare 8M Urea/40m HEPES aliquots, store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>High-resolution mass spectrometer</td>
			<td>ThermoFisher Scientific</td>
			<td>726042</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide&#160; (IAA)</td>
			<td>Fisher Scientific&#160;</td>
			<td>AAA1471514</td>
			<td>Prepare 0.55M stock of IAA aliquots and store at&#160; -20&#176;C. Avoid light exposure&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>25030081</td>
			<td>Thaw out, prepare aliquots and stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>LoBind Microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>13-698-794</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant&#160;</td>
			<td>https://www.maxquant.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR strip tubes</td>
			<td>VWR</td>
			<td>76318-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin, Amphotericin</td>
			<td>Sigma Aldrich</td>
			<td>A5955</td>
			<td>Thaw out, prepare aliquots and stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>Perseus&#160;</td>
			<td>https://www.maxquant.org/Perseus/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate-buffered solution (PBS)&#160;</td>
			<td>Bioshop&#160;</td>
			<td>PBS408</td>
			<td>Store at room temperature&#160;</td>
		</tr>
		<tr>
			<td>Probe sonicator&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>100-132-894</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (PIC) tablet&#160;</td>
			<td>Sigma Aldrich</td>
			<td>5892791001</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>11875093</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulphate (SDS)</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>J18220.A1</td>
			<td>prepare 2% SDS solution</td>
		</tr>
		<tr>
			<td>SpeedVac Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>07-748-15</td>
			<td></td>
		</tr>
		<tr>
			<td>STAGE tipping centrifuge</td>
			<td>Sonation</td>
			<td>STC-V2</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>ThermoFisher Scientific</td>
			<td>85183</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris HCL pH 8.5</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin/LysC</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>A40009</td>
			<td>aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>VWR</td>
			<td>97043-960</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea&#160;</td>
			<td>Bioshop&#160;</td>
			<td>URE001.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

This video demonstrates peptide purification through the STAGE Tip procedure. A miniature column containing resin beads bonded to long hydrocarbon chains facilitates hydrophobic interaction-based purification. Upon loading a pre-digested peptide sample into the column, the peptide fragments bind to the resin beads, allowing impurities to pass through. An elution buffer that disrupts the hydrophobic interaction is applied to elute the peptide fragments.

1. Purification and Desalting of Peptides

	Pack three layers of C18 resin in a 200 &#956;l pipette tip for each peptide sample to construct a STop And Go ...

Take a STop And Go Extraction or STAGE Tip, a miniaturized purification column.
The tip contains small silica beads bonded to long hydrocarbon chains for hydrophobic interaction-based peptide purification.
Wash with buffers containing a high percentage of acetonitrile, removing resin impurities.
Introduce a binding buffer and centrifuge, equilibrating the column for optimal peptide binding.
Load a pre-digested peptide sample obtained from differentiated neutrophil-like cells, containing peptide fragments and impurities, including salts.
Centrifuge to pass the sample through the column. The peptide fragments bind to the hydrocarbon chains via hydrophobic interactions, while the salts are removed.
Add the binding buffer and centrifuge, removing the remaining impurities.
Introduce the high acetonitrile-containing buffer for elution.
Using a syringe, pass the buffer through the column. Acetonitrile binds to the hydrocarbon chains, displacing the peptide fragments that elute with the buffer. 
Perform proteomic profiling to identify the purified peptides.

338 Aufrufe. Ein STAGE-Spitzenverfahren zur Entsalzung und Reinigung von Peptiden

Serie: Ein STAGE-Spitzenverfahren zur Entsalzung und Reinigung von Peptiden - Experiment

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Ein Tailored HPLC Reinigungsprotokoll, dass die Renditen hochreine Amyloid Beta 42 und Amyloid Beta 40 Peptides, Fähig zur Oligomerbildung

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Forschung

JoVE Journal

Biochemie

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.

Nachweis von Protein-Ubiquitinationsstellen durch Peptidanreicherung und Massenspektrometrie

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

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

A STAGE Tip Procedure for Desalting and Purification of Peptides

Transkript

A STAGE Tip Procedure for Desalting and Purification of Peptides