Name: Video: Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions - Concept
Uploaded: 2023-04-30T14:00:50.000+00:00
Duration: 2 min 13 s
Description: 1.0K Views. Source: Bezstarosti, K. et al. Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. J. Vis. Exp. (2020) This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein.

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1. Extraction of Ubiquitinylated Peptides
<ol>
	<li>Use high pH reverse-phase (RP) C18 chromatography with polymeric stationary phase material (300 &#197;, 50 &#181;M; see Table of Materials) loaded into an empty column cartridge to fractionate the tryptic peptides. The stationary phase bed size must be adjusted to the amount of protein digest to be fractionated. Prepare an empty 6 mL column cartridge (see Table of Materials) filled with 0.5 g of stationary phase material for ~10 mg of protein digest. The protein digest to stationary phase ratio should be approximately 1:50 (w/w).</li>
	<li>Load the peptides onto the prepared column and wash the column with approximately 10 column volumes of 0.1% TFA, followed by approximately 10 column volumes of H2O.</li>
	<li>Elute the peptides into three fractions with 10 column volumes of 10 mM ammonium formate solution (pH = 10) with 7%, 13.5%, and 50% acetonitrile (AcN), respectively. Lyophilize all fractions to completeness.</li>
	<li>Use ubiquitin remnant motif (K-&#949;-GG) antibodies conjugated to protein A agarose beads for the immunoenrichment of diGly peptides. Because the exact amount of antibody per batch of beads is proprietary information and not disclosed by the manufacturer, it is recommended to use the same definition for a batch of beads as the manufacturer does in order to avoid confusion. Wash one batch of these beads 2x with PBS and split the bead slurry into six equal fractions. See&#160;Figure 1&#160;for a detailed experimental scheme.</li>
	<li>Dissolve the three peptide fractions collected in step 1.3 in 1.4 mL of a buffer composed of 50 mM MOPS, 10 mM sodium phosphate, and 50 mM NaCl (pH = 7.2), and spin down the debris.</li>
	<li>Add the supernatants of the fractions to the diGly antibody beads and incubate for 2 h at 4 &#176;C on a rotator unit. Spin down the beads and transfer the supernatant to a fresh batch of antibody beads and incubate again for 2 h at 4 &#176;C.</li>
	<li>Store the supernatants for subsequent global proteome (GP) analysis.</li>
	<li>Transfer the beads from every fraction into a 200 &#181;L pipette tip equipped with a GF/F filter plug to retain the beads. Put the pipette tip with the beads into a 1.5 mL microcentrifuge tube equipped with a centrifuge tip adapter. Wash the beads 3x with 200 &#181;L of ice-cold IAP buffer and subsequently 3x with 200 &#181;L of ice-cold Milli-Q H2O. Spin down the column at 200 x&#160;g&#160;for 2 min before every wash step but be careful not to let the column run dry. Elute the peptides using 2 cycles of 50 &#181;L of 0.15% TFA.</li>
	<li>Desalt the peptides using a C18 stage tip (essentially a 200 &#181;L pipette tip with two C18 disks) and dry them to completeness using vacuum centrifugation.</li>
</ol>

<table><tbody><tr><td>Lysyl Endopeptidase(LysC)</td><td>Wako Pure Chemicals</td><td>129-02541</td><td></td></tr><tr><td>NanoLC oven</td><td>MPI design, MS Wil GmbH</td><td></td><td></td></tr><tr><td>N-Lauroylsarcosine sodium salt</td><td>Sigma-Aldrich</td><td>L-5125</td><td></td></tr><tr><td>Orbitrap Fusion Lumos mass&lt;br/&gt; spectrometer</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>PLRP-S (300 &amp;Aring;, 50 &amp;micro;m) polymeric&lt;br/&gt; reversed phase particles</td><td>Agilent Technologies</td><td>PL1412-2K01</td><td></td></tr><tr><td>PTMScan Ubiquitin Remnant Motif&lt;br/&gt; (K-&amp;epsilon;-GG) Kit</td><td>Cell Signaling Technologies</td><td>5562</td><td></td></tr><tr><td>Sep-Pak tC18 6 cc Vac Cartridge</td><td>Waters</td><td>WAT036790</td><td>Remove the tC18 material from the&lt;br/&gt; cartridge before filling the cartridge&lt;br/&gt; with PLRP-S</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>30970</td><td></td></tr><tr><td>Tris-base</td><td>Sigma-Aldrich</td><td>T6066</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Trypsin, TPCK Treated</td><td>ThermoFisher</td><td>20233</td><td></td></tr></tbody></table>

immunoaffinity based extraction of ubiquitinylated peptides: a technique to selectively extract ubiquitin tagged peptides from purified peptide fractions

Source: Bezstarosti, K. et al. <a href="https://www.jove.com/v/59079/detection-protein-ubiquitination-sites-peptide-enrichment-mass">Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. J. Vis. Exp.</a> (2020)
This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein.

<img alt="Figure 1" src="/files/ftp_upload/20622/20622_Figure1.jpg" /> 
Figure 1: Experimental overview.&#160;(A) Overview of the experimental approach. Samples were prepared, trypsinized, and fractionated into three fractions using reverse-phase chromatography with high pH elution. One batch of commercial &#945;-diGly peptide antibody beads was split into six equal fractions and the three peptide fractions were then loaded on three of the bead fractions. The diGly peptides were immunopurified, eluted, ...

Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions

Source: Bezstarosti, K. et al. Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. J. Vis. Exp. (2020)
This video ...

Ubiquitination of proteins is characterized by the addition of ubiquitin - a regulatory mini-protein - via its di-glycine residues to the lysine residues of the substrate protein. This generates a lysine-glycine-glycine linkage between the substrate protein and ubiquitin.
To isolate ubiquitin-tagged peptides, begin by taking a peptide mix.
Treat these peptides with an endonuclease enzyme cocktail. During treatment, the enzymes Lys-C and trypsin efficiently cleave the ubiquitinylated peptides at the arginine residue, resulting in peptide fragments containing a signature di-glycine motif.
Add these peptide fragments to a slurry of antibody-conjugated hydrogel beads.
These antibodies contain a di-glycine recognition site, which binds exclusively to the ubiquitinylated peptides, forming a peptide-bead complex.
Centrifuge to pelletize ubiquitinylated peptide-bead complexes. Remove the unbound non-ubiquitinylated peptide-containing supernatant.
Load the complexes onto a filter assembly. Centrifuge to remove the buffer and trap peptide-conjugated beads.
Add an acidified reagent that lowers the pH, causing disengagement of peptides from the beads.
Recover the free peptides from the filtrate. Load them onto a fresh filter with a hydrophobic matrix. The peptides retain in the matrix while acidic salts get washed away.
Using a suitable solvent, detach the peptides.
Transfer the ubiquitinylated peptides to a fresh tube and dry them to remove any traces of solvent.

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1.0K Views. Source: Bezstarosti, K. et al. Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. J. Vis. Exp. (2020) This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein. 

Video: Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions - Concept

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Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions

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Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis