Proteins were digested in the gel as previously described with minor modifications (Ishikawa et al., 2005 (link)). The target protein was stained with Quick CBB (Fujifilm Wako Chemical, Tokyo, Japan), cut from the gel, de-stained in 50% (vol/vol) acetonitrile in water, dehydrated in 100% acetonitrile, and dried in a vacuum desiccator. ProteoExtract All-in-One Trypsin Digestion Kit (Merck-Millipore, Burlington, MA, USA) was used for in-gel tryptic digestion. The peptide mixtures were applied to a liquid chromatography–quadrupole time-of-flight mass spectrometry system (QSTAR Elite; AB Sciex, Framingham, MA, USA) equipped with a Cadenza CD-C18 2.0×150 mm column (Imtakt, Kyoto, Japan) and eluted in 75 min at a flow rate of 0.2 mL/min with a 3%–40% linear gradient of acetonitrile containing 0.1% formic acid. The data of peptide mass fingerprinting were searched against the protein database generated from the BF-1 genome.
Proteoextract all in one trypsin digestion kit
Manufactured by Merck Group
Sourced in United States
The ProteoExtract All-in-One Trypsin Digestion Kit is a laboratory product designed for the proteolytic digestion of proteins. It contains all the necessary reagents and components for efficient protein digestion using trypsin, a widely used enzyme in protein analysis workflows.
Automatically generated - may contain errors
Lab products found in correlation
Pepswift monolithic nano column, by Thermo Fisher Scientific (2 mentions)
Peaks studio 8, by Bioinformatics Solutions (2 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
C18 ziptip, by Merck Group (2 mentions)
Qstar elite, by AB Sciex (1 mentions)
4 protocols using proteoextract all in one trypsin digestion kit
1
Protein Identification via Mass Spectrometry
2
Quantification of Mutant TnT-I79N in hiPSC-CMs
3
Proteome Analysis of A. lumbricoides Worm
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5 μL of A. lumbricoides extract (1000 μg/mL) were digested with the ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, MA, USA) and desalted using C18ZipTips (EMD Millipore, Billerica, MA, USA). Resulting peptides were separated by reverse-phase nano-high performance liquid chromatography (HPLC, Dionex Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany, column: PepSwift Monolithic Nano Column, 100 μM × 25 cm, Dionex). The column was developed with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 μL/min at 55 °C). The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2 kV. For peptide assignment, a top 12 MS/MS method was used with the normalized fragmentation energy set to 27%. Proteins were identified with PEAKS Studio 8 (Bioinformatics Solutions, Waterloo, Canada), using the UniProt (SwissProt/TrEMBL) sequence database. For the identification of post-translational modifications and amino acid exchanges, the PTM and Spider modules of PEAKS Studio were used. Only peptides with high confidence scores (−10lgp ≥ 35, corresponding to false discovery rate (FDR) < 0.5%) were considered in the database searches.
4
Proteomic Profiling of A. lumbricoides
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A. lumbricoides extract (1000 µg/mL) was digested using the ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, MA, USA) and desalted using C18ZipTips (EMD Millipore, Billerica, MA, USA). Resulting peptides were separated by reverse-phase nano-high performance liquid chromatography (HPLC, Dionex Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany, column: PepSwift Monolithic Nano Column, 100 µM × 25 cm, Dionex). The column was developed with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 µL/min at 55 °C. The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2 kV. For peptide assignment, a top 12 MS/MS method was used with the normalized fragmentation energy set to 27%. The protein was identified with PEAKS Studio 8 (Bioinformatics Solutions, Waterloo, ON, Canada) using the UniProt (SwissProt/TrEMBL) sequence database. Only peptides with high confidence scores (−10lgp ≥ 35, corresponding to false discovery rate (FDR) < 0.5%) were considered in the database searches [18 (link)]. Abundance of Al-CPI in the extract was calculated based on the “Area” parameter from the summarized proteomic search results [23 (link)].
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