Samples were re-suspended in 350 μL 8 M urea/ 100 mM Tris HCl pH 8.5 after TCA/acetone precipitation and phenol extraction. Briefly, samples were reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) for 30 min at room temperature and alkylated with 10 mM iodoacetamide for 30 min at room temperature in the dark. Then, proteins were firstly digested for 5 h at 37°C with 250 ng rLys-C Mass Spec Grade (Promega, Madison, USA). Samples were then diluted 4-fold with 100 mM Tris HCl pH 8.5 to reach a concentration of 2M urea and then re-incubated overnight at 37°C with 1 μg Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA). A second incubation with the same amount of trypsin (5 h at 37°C) was performed to ensure a complete digestion. Digestion was stopped by adding formic acid to 5% final concentration and peptides were desalted and concentrated on Sep-Pak C18 SPE cartridge (Waters, Milford, MA, USA) according to manufacturer’s instructions.
Rlys c mass spec grade
Manufactured by Promega
Sourced in United States
The RLys-C Mass Spec Grade is a laboratory reagent designed for use in mass spectrometry applications. It serves as a chaotropic lysis buffer for the extraction and solubilization of proteins from biological samples.
Automatically generated - may contain errors
6 protocols using rlys c mass spec grade
1
Protein Extraction and Digestion Protocol
2
Protein Extraction and Digestion Protocol
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One milligram of protein extract was solubilized in 8 M urea, 100 mM Tris HCl pH 8.5, then disulfide bonds were reduced with 5 mM Tris(2-carboxyethyl) phosphine (TCEP) for 30 min and alkylated with 10 mM iodoacetamide for 30 min at room temperature (RT) in the dark. Protein samples were then incubated with rLys-C Mass Spec Grade (Promega, Madison, WI, USA) for 5 h at 30 °C for the first digestion (ratio 1:50). Then samples were diluted below 2 M urea with 100 mM Tris HCl pH 8.5 and Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) was added for the second digestion overnight at 37 °C (ratio 1:100). Digestion was stopped by adding 5% of formic acid (FA) and peptides were desalted and concentrated on Sep-Pak light tC18 SPE cartridge (Waters, Milford, MA, USA) according to manufacturer’s instructions. Peptides were eluted using 50% acetonitrile (ACN), 0.1% FA. Purified peptides were lyophilized and kept at −80 °C until further used.
3
Protein Extraction and Digestion Protocol
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Conidia were incubated in formic acid for 2 h as described above. Preliminary experiments showed that the same proteins were extracted by 10 min or 2 h incubation times. Dried samples were re-suspended in 100 µl 8 M urea, 100 mM Tris-HCl pH 8.5. Briefly, samples were reduced with 5 mM TCEP for 30 min and alkylated with 10 mM iodoacetamide for 30 min at room temperature in the dark. Protein samples were then incubated with 250 ng rLys-C Mass Spec Grade (Promega, Madison, WI, USA) for 5 h at 37 °C for the first digestion. Samples were then diluted to 2 M urea with 100 mM Tris HCl pH 8.5 and 500 ng sequencing grade modified trypsin (Promega, Madison, WI, USA) was added for the second digestion overnight at 37 °C. A second incubation with the same amount of trypsin (5 h at 37 °C) was performed to ensure a complete digestion. Digestion was stopped by adding formic acid -and peptides were desalted and concentrated on a Sep-Pak C18 SPE cartridge (Waters, Milford, MA, USA) according to manufacturer’s instructions.
4
Protein Reduction, Alkylation, and Digestion
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Proteins were reduced using 5 mM TCEP for 30 min at room temperature. Alkylation of the reduced disulfide bridges was performed using 10 mM iodoacetamide for 30 min at room temperature in the dark. Proteins were then digested in two steps, first with 250 ng r-LysC Mass Spec Grade (Promega) for 4 h at 30 °C then samples were diluted below 2 M urea with 100 mM Tris HCl pH 8.5 and 500 ng Sequencing Grade Modified Trypsin was added for the second digestion overnight at 37 °C. Proteolysis was stopped by adding formic acid (FA) at a final concentration of 5%. The resulting peptides were cleaned using AssayMAP C18 cartridges on the AssayMAP Bravo platform (Agilent) according to the manufacturer’s instructions. Peptides were concentrated to dryness and resuspended in 2% acetonitrile (ACN) and 0.1% FA just prior to LC-MS injection.
5
Separating Type I Collagen Chains
6
Workflow for Protein Reduction, Alkylation, and Tryptic Digestion
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Proteins were reduced using 5 mM TCEP for 30 min at room temperature. Alkylation of the reduced disulfide bridges was performed using 10 mM iodoacetamide for 30 min at room temperature in the dark. Proteins were then digested in two steps, first with 1 µg r-LysC Mass Spec Grade (Promega) for 4 hr at 30°C and then samples were diluted below 2 M urea with 100 mM Tris HCl pH 8.5 and 1 µg Sequencing Grade Modified Trypsin was added for the second digestion overnight at 37°C. Proteolysis was stopped by adding formic acid (FA) at a final concentration of 5%. The peptide samples were cleaned using C18 cartridges (Agilent Technologies) in the automated AssayMAP Bravo Platform. The protocol for peptide cleanup included in the platform was used as a scaffold using the following settings. Cartridges were primed with 100 μL of 100% acetonitrile (ACN) at 300 μL/min. Equilibration was done with 50 μL of 0.1% FA at 10 μL/min. The sample was loaded at 5 μL/min. Cup wash (25 μL) and the internal cartridge wash (50 μL at 10 μL/min) were performed with 0.1% FA. Peptides were eluted with 50 μL of 50% ACN/0.1% FA at 5 μL/min. Samples were dried and stored at −80°C until further use. Peptides were resuspended in 2% acetonitrile (ACN)/0.1% FA prior to LC-MS injection.
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