HPLC purified peptide was incubated in the presence of several enzymes in parallel reactions. The enzymes used were endoproteinase GluC (New England BioLabs, Ipswich, MA), leucine aminopeptidase (Sigma-Aldrich, St. Louis, MO), carboxypeptidase Y (Sigma-Aldrich, St. Louis, MO), trypsin (Thermo Fischer, Waltham, MA), and proteinase K (Promega, Madison, WI). The C3 peptide was incubated at 37°C for all reactions unless otherwise specified. The endoproteinase GluC reaction had a final concentration of 1X GluC reaction buffer (New England BioLabs, Ipswich, MA). The leucine aminopeptidase (Sigma-Aldrich, St. Louis, MO) reaction used a final concentration of 50 mM NaPO4 (pH 7.2). Cell grade trypsin/EDTA (0.25%) (Thermo Fischer, Waltham, MA) used deionized H2O for its reaction. carboxypeptidase Y (Sigma-Aldrich, St. Louis, MO) reaction used a final concentration of 50 mM 2-[N-morpholino]ethanesulfonic acid (MES) pH 6.75, this reaction was carried out at 25°C. Enzyme concentrations were made at a ratio enzyme:substrate 1:50–1:100. Proteolytic reactions occurred over 4 h incubations or overnight. Peptide degradation was determined using MALDI-TOF as previously described. Recombinant SdrC, a purified protein was used as a control.
Endoproteinase gluc
Manufactured by New England Biolabs
Sourced in United States
Endoproteinase GluC is a proteolytic enzyme that cleaves peptide bonds on the carboxyl side of glutamic acid residues. It is commonly used in protein analysis and purification workflows to generate specific peptide fragments for downstream applications such as mass spectrometry and N-terminal sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
Coomassie brilliant blue, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslcnano hplc system, by Thermo Fisher Scientific (2 mentions)
Orbitrap elite mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Tris carboxyethyl phosphine, by Hampton Research (2 mentions)
Glu c, by Roche (2 mentions)
Endoproteinase asp n, by Roche (2 mentions)
α cyanohydroxycinnamic acid, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Acetonitrile, by Carl Roth (1 mentions)
Hitrap con a 4b column, by GE Healthcare (1 mentions)
Amicon ultra centrifugal concentrator, by Merck Group (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
Hiload 16 60 superdex 200 pg column, by GE Healthcare (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Anti gfp antibody, by Abcam (1 mentions)
Maldi tof tof mass spectrometer, by Bruker (1 mentions)
Maldi matrix 2 5 dihydroxybenzoic acid, by Bruker (1 mentions)
17 protocols using endoproteinase gluc
1
Peptide Degradation by Common Proteases
2
Mass Spectrometry Proteomics Reagents
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Acetonitrile (AcN), methanol (MeOH), dichlormethane,
trifluoroacetic acid (TFA), and H2O were purchased as HPLC
or LC-MS grade from Carl Roth (Karlsruhe, Germany). Dithiothreitol
(DTT), α-cyano-hydroxy cinnamic acid, trypsin (sequencing grade),
and iodoacetamide (IAA) were purchased as Bioultra grade from Sigma-Aldrich
(Sigma-Aldrich, Vienna, Austria). Endoproteinase Glu C (sequencing
grade) was purchased from New England Biolabs (Ipswich, MA).
trifluoroacetic acid (TFA), and H2O were purchased as HPLC
or LC-MS grade from Carl Roth (Karlsruhe, Germany). Dithiothreitol
(DTT), α-cyano-hydroxy cinnamic acid, trypsin (sequencing grade),
and iodoacetamide (IAA) were purchased as Bioultra grade from Sigma-Aldrich
(Sigma-Aldrich, Vienna, Austria). Endoproteinase Glu C (sequencing
grade) was purchased from New England Biolabs (Ipswich, MA).
3
Purifying and Complexing PfRH5ΔNL from Drosophila
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PfRH5ΔNL was purified from Drosophila S2 culture supernatant using C-tag affinity chromatography and glycosylated contaminants were removed by a subsequent lectin chromatography step with a HiTrap ConA 4B column (GE Healthcare). Disordered regions were trimmed by an overnight incubation at 20°C with endoproteinase gluC (New England Biolabs) at a final concentration of 1 μg/mL. Fab fragments were generated by papain digestion using a Pierce Fab Preparation Kit (Thermo Fisher Scientific) following the manufacturer’s recommendations. Complexes were prepared by mixing each Fab fragment with PfRH5ΔNL at a 1:1 molar ratio and were methylated with 1 M ABC (Borane dimethylamine complex) and 1 M formaldehyde (both Sigma-Aldrich) (Walter et al., 2006 (link)). The methylated complexes were subjected to SEC on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) at 4°C in 20 mM Tris pH 7.4, 150 mM NaCl. The complex-containing fractions were pooled and concentrated using an Amicon® ultra centrifugal concentrator (Millipore) with a molecular weight cut-off of 30 kDa.
4
VSG3 Proteome Analysis by Mass Spectrometry
5
GFP-mCherry Protein Analysis
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HEK 293T cells grown on 15 cm cell culture plates were transfected with 40 μg GFP–mCherry plasmid (WT, GAA, or GAG) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. Cells were collected after 48 h for immunoprecipitation with anti-GFP antibody (Abcam or Santa Cruz). Purified proteins were eluted from protein A beads (Invitrogen) using 0.1 M glycine, pH 3, and analyzed by SDS-PAGE. 5 µg of GFP–mCherry proteins were digested with 50 ng/µL endoproteinase Glu-C (New England Biolabs) at 37°C for 24 h. Digested peptides were mixed with MALDI matrix 2,5-dihydroxybenzoic acid (Bruker) and analyzed in an MALDI-TOF/TOF mass spectrometer (Bruker).
6
Mass Spectrometry Analysis of NPC1-EGFP
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Purified NPC1–EGFP-containing membranes were subjected to reduction and alkylation, digestion with trypsin or endoproteinase GluC (New England Biolabs) similar to that described previously (Champion et al., 2012 (link); Williams et al., 2017 (link)). Samples were desalted by ZipTips (Millipore, Billerica, MA) according to the manufacturer's instructions and separated on a nanoAcquity UHPLC system (Waters Billerica, MA) prior to MS/MS analysis. Liquid chromatography (LC)/MS-MS analysis was performed on an amaZon ion trap instrument (Bruker Daltronics, Billerica, MA) running in data-dependent acquisition mode. Acquisition and processing were performed in the Mass Spectrometry and Proteomics Facility (MSPF) at the University of Notre Dame. Spectra were converted to .mgf files (MS/MS peak lists) using Data Analysis (Bruker) and subjected to database search with Mascot against the human UniProt database (downloaded 15 October 2009). MS tolerances were set to ±1.5 Da for MS1 and 1.7 Da for MS2. Owing to the small search space, traditional false discovery rate was not employed as it is not accurate for small-n datasets. A Mascot score of 40 and two peptides uniquely matching were used for ID cutoff.
7
Comprehensive Synthesis of Bioactive Compounds
8
GST-HMG-C-clamp DNA Binding Assay
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20 µl reactions containing 3–6 µM GST-HMG-C-clamp and 20× of the indicated DNA oligonucleotide were incubated for 5 min on ice and 15 min at room temperature. The buffer was the same as used for EMSA but without poly-dI*dC. Protease was then added (for partial proteolytic digestion) or not (for reverse EMSA) at a final concentration of 5–50 ng/µl for chymotrypsin (Roche) or 50–150 ng/µl for endoproteinase Glu-C (New England Biolabs). The mixture was incubated at 25°C for 2.5–3 hours. Then the digested product was loaded onto 16% tricine SDS-PAGE gel [66] (link), and the undigested mixture was loaded onto 6% native PAGE-gel. After running, the gels were silver stained as previously described [67] (link).
9
Hydrolysis of ThiS-COSH by TfuA in [18O]-H2O
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MtThiS-COSH was lyophilized to remove H2O and resuspended in [18O]-H2O. For the hydrolysis reaction, 5 μM of MtTfuA (1 μL) was incubated with 45 μM MtThiS-COSH in [18O]-H2O (9 μL) at room temperature for 1 h. The reaction was stopped by the addition of endoproteinase GluC (NEB) at the final concentration of 5 ng/μL and incubation at 37 °C for 16 h. The resulting fragment of MtThiS-COSH was analyzed by MALDI-TOF-MS (desalted using a ZipTip and eluted into 70% aq. MeCN) with CHCA as the matrix. The variants were tested the same way as wild-type MtTfuA using the same batch of ThiS-COSH.
10
VSG3 Proteome Analysis by Mass Spectrometry
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Purified VSG3 (50 μg in 25 μl 10 mM sodium phosphate, pH 8) was reduced (10 mM DTT, 85 °C, 20 min), S-alkylated (25 mM IAA, 1h, RT in the dark), diluted with an equal volume of 2 x GluC buffer and digested with 1:25 (w/w) endoproteinase GluC (New England BioLabs) for 24 h, 37 °C, with shaking. The GluC fragments were separated using NuPAGE 4-12% Bis-Tris protein gels (Invitrogen), stained with Coomassie Brilliant Blue (Thermo Scientific) and the 17 kDa fragment was excised and subjected to in-gel trypsin digestion for LC-MS/MS analysis.
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