Samples (omvPV or wcPV) were diluted in a denaturation buffer containing 1 M guanidine hydrochloride (Gnd-HCl) and 50 mM triethylammonium bicarbonate, pH 8.5, to a final protein concentration of 0.2 mg/mL. Reduction and alkylation of disulfide bridges was performed with 5 mM TCEP (Thermo, Rockford, IL, USA) (1 h at 55 °C) and 9.4 mM iodoacetamide (30 min at RT in the dark). Proteins were digested with 0.5 µg endoproteinase Lys-C (Roche, Mannheim, Germany) followed by incubation (4 h, 37 °C). Subsequently, digests were incubated (ON, 37 °C) with 1 μg trypsin (Promega, Madison, WI, USA). Solid-phase extraction was performed to remove excess reagents using C18 Sep-pack cartridges (Waters, Milford, MA, USA) according to the manufacturer’s protocol. Peptides were dried in a vacuum concentrator. Peptides were dissolved in 100 µL formic acid/DMSO/water (0.1/5/94.9% v/v) for LC–MS/MS analysis.
Endoproteinase lys c
Manufactured by Roche
Sourced in Germany
Endoproteinase Lys-C is a proteolytic enzyme that cleaves peptide bonds specifically on the carboxyl side of lysine residues in proteins. It is used in protein sequencing and analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Roche (5 mentions)
Benzonase, by Merck Group (4 mentions)
Protease inhibitor tablet, by Roche (3 mentions)
Modified trypsin, by Promega (3 mentions)
Trypsin, by Promega (2 mentions)
293 expi cells, by Thermo Fisher Scientific (2 mentions)
293f cells, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 agarose, by Merck Group (2 mentions)
C18 sep pack cartridges, by Waters Corporation (1 mentions)
C18 resin, by Phenomenex (1 mentions)
Zorbax extend c18 analytical column, by Agilent Technologies (1 mentions)
2d nanolc, by AB Sciex (1 mentions)
Ltq orbitrap, by Thermo Fisher Scientific (1 mentions)
2 chloroacetamide, by Merck Group (1 mentions)
Pcdna5 frt to vector, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Oasis hlb, by Waters Corporation (1 mentions)
Anti transgelin, by Abcam (1 mentions)
16 protocols using endoproteinase lys c
1
Protein Denaturation and Digestion Protocol
2
Protein Fractionation and Digestion
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The samples were reduced using 5 mM TCEP for 20 min, and alkylated by 50 mM chloroacetamide for 30 min in the dark at room temperature. The proteins were first digested with endo-proteinase LysC (Roche, 11047825001, 1:100 w/w) at 35 °C for an hour. The mixture was then diluted to 2 M urea with tryptic digestion buffer (100 mM Tris-HCl, 1 mM CaCl2, pH 8.5) and the proteins were further digested by trypsin (Thermo, PRV5113, 1:20 w/w) for 20 hours at 35 °C. The resulting peptides from total crude lysate and soluble fractions were purified by STop-And-Go Extraction (STAGE) high-capacity tips using C18 resin (Phenomenex)50 (link).
Roughly 100 μg of peptides of each sample was fractionated by offline high pH reversed-phase chromatography using a Zorbax Extend-C18 analytical column, 5 μm, 4.6 × 50 mm (Agilent), on a 64 minute gradient (followed by a 21 minute equilibration with buffer A) with a 50 μL/min flow rate, where Buffer A contained 5 mM NH4HCO2, pH 10 and 2% acetonitrile and Buffer B contained 5 mM NH4HCO2, pH 10 and 90% acetonitrile. Ninety-six fractions were collected at 40 seconds/fraction. The resulting fractions were pooled in a non-contiguous manner, as previously described51 (link). The fractionated samples were pooled into 9 fractions.
Roughly 100 μg of peptides of each sample was fractionated by offline high pH reversed-phase chromatography using a Zorbax Extend-C18 analytical column, 5 μm, 4.6 × 50 mm (Agilent), on a 64 minute gradient (followed by a 21 minute equilibration with buffer A) with a 50 μL/min flow rate, where Buffer A contained 5 mM NH4HCO2, pH 10 and 2% acetonitrile and Buffer B contained 5 mM NH4HCO2, pH 10 and 90% acetonitrile. Ninety-six fractions were collected at 40 seconds/fraction. The resulting fractions were pooled in a non-contiguous manner, as previously described51 (link). The fractionated samples were pooled into 9 fractions.
3
Production and Purification of 10E8 Antibody
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Transient expression of the 10E8 antibody was undertaken in 293F cells or 293 Expi cells per the manufacturer’s instructions (Life Technologies) by co-transfection of equal amounts of the 10E8 heavy and light chain plasmids, as previously described66 (link). 10E8 IgG was purified by capture with Protein A sepharose (Pierce) followed by elution in low pH (Pierce) with adjustment of eluate pH with Tris-Cl pH 8.0. The 10E8 Fab was prepared using endoproteinase Lys-C (Roche) digestion, as described66 (link). The LysC protease was added at concentrations of 1:1,000 to 1:10,000 and the digestion undertaken at 37°C for 4–6 h. Digestion reactions were stopped with protease inhibitor tablets (Roche) and cleaved products were run over a Protein A column to segregate the Fc fragment. 10E8 Fab was then subjected to cation exchange (Mono S) and size-exclusion (S200) chromatography, followed by dialysis of peak fractions into 150 mM NaCl, 2.5 mM Tris, pH 7.5, 0.02% NaN3.
4
Production and Purification of 10E8 Antibody
5
LEDGF/p75 Interactome Purification
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LEDGF/p75 was doxycycline inducibly expressed in HEK293FRT cells following the manufacturer’s instructions (Thermo Fisher Scientific, R75007), and the nuclear extract was prepared according to the Dignam method. LEDGF/p75 bound proteins were purified on anti-Flag (M2) agarose beads in the presence of Benzonase (Sigma, E1014), separated on SDS–polyacrylamide gel electrophoresis gel, and visualized by silver staining. Trichloroacetic acid–precipitated protein mixtures from the Flag purifications were digested with endoproteinase Lys-C and trypsin (Roche, 11058533103) and analyzed by MudPIT as previously described (40 (link)).
6
Protein Purification and Mass Spectrometry
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TCA-precipitated protein pellets were solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37°C overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37°C using trypsin (Roche) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The digested size exclusion eluates were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. The digested Not and control FLAG-IP eluates were analyzed on an LTQ-Orbitrap (Thermo) coupled to an Eksigent NanoLC-2D. In both cases, a fully automated 10-step chromatography run was carried out. Each full MS scan (400–1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to one both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100.
7
Nuclear Protein Purification and Mass Spec
8
Luciferase Protein Sequencing by Mass Spectrometry
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The full-length luciferase readthrough products resolved on SDS-polyacrylamide gel were excised after silver staining of the gel and the destained gel slices were subjected to endoproteinase Lys-C (Roche Diagnostics) digestion followed by LC-MS/MS analyses as described previously (Roy et al. 2015 (link), 2016 (link)). Raw data files were subjected to database searching with Mascot Server (version 2.4) against human SwissProt index that contain the sequences of all 20 potential mutations of the fLuc protein at each position investigated. The relative abundance of each amino acid at position 20 of luciferase was calculated by adding the corresponding precursor intensity of individual endo Lys-C peptide containing the codon 20 to yield a total peptide abundance that was then used to calculate the percentage of insertion of each amino acid (Roy et al. 2015 (link), 2016 (link)).
9
Streptavidin/FLAG Affinity Protein Purification
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TCA-precipitated protein samples from streptavidin affinity purifications or FLAG immunoprecipitations were solubilized in 30 µl of freshly made 0.1 M Tris-HCl, pH 8.5, 8 M urea, 5 mM TCEP (Tris [2-Carboxylethyl]-Phosphine Hydrochloride, Pierce/ Thermo Fisher Scientific). After 30 min at room temperature, freshly made 0.5 M 2-Chloroacetamide (Sigma-Aldrich) was added to a final concentration of 10 mM, and the samples were left at room temperature for another 30 min in the dark. Endoproteinase Lys-C (Roche) was first added at an estimated 1:100 (wt/wt) enzyme to protein ratio, for at least 6 hr at 37°C. Urea was then diluted to 2 M with 0.1 M Tris-HCl, pH 8.5, CaCl2 was added to 0.5 mM, and modified trypsin (Promega; Madison, WI), 1:100 (wt/wt), was added for over 12 hr at 37°C. All enzymatic digestions were quenched by the addition of formic acid to 5% final concentration.
10
Nuclear Protein Purification and Mass Spec
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Anti-Flag M2 agarose was purchased from Sigma. Nuclear extract preparation and Flag affinity purifications were performed in the presence of Benzonase (Sigma). Trichloroacetic acid-precipitated protein mixtures from the Flag purifications were digested with endoproteinase Lys-C and trypsin (Roche) and analyzed by MudPIT (Lin et al., 2010 (link)).
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