An in vitro ΔMKD1 kinase assay using MKKs was performed as previously described (Nishihama et al., 2001 (link)). The assay used 200 ng protein. An in-gel kinase assay was performed using 20 µg total proteins as described elsewhere (Nishiuchi et al., 2006 (link)). An immunoprecipitation kinase assay was performed using anti-MPK4 (a gift from Y. Machida, Nagoya University) and anti-MPK6 (Sigma-Aldrich) antibodies as previously described (Nishiuchi et al., 2006 (link)). The phosphorylation sites were determined by LC-MALDI analysis. The phosphorylated MKK1 and MKK5 proteins were digested by chymotrypsin (Roche). The peptides were analysed using a 4800 Plus MALDI TOF/TOFTM Analyzer (AB Sciex). tandem mass spectrometry (MS/MS) data were evaluated by comparing amino acid substitutions and modifications against the NCBI database using the Paragon algorithm (Shilov et al., 2007 (link)) of the ProteinPilot™ v2.0 software (AB Sciex).
Proteinpilot v2
Manufactured by AB Sciex
ProteinPilot v2.0 is a software application developed by AB Sciex for the analysis and identification of proteins from mass spectrometry data. It provides automated protein identification and quantification capabilities.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using proteinpilot v2
1
In vitro ΔMKD1 kinase assay
2
Quantitative Proteome Profiling by IPG-IEF
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Labeled peptides were fractionated and purified by immobilized-pH-gradient isoelectric focusing (IPG-IEF), as previously described 44 (link) . Purified peptide fractions were reconstituted in solvent A. The peptides were electrosprayed using a nanoelectrospray ionization source at an ion spray voltage of 2300 eV and analyzed by a NanoLC-ESI-Triple TOF 5600 system (AB Sciex). The mass spectrometer was set in the positive ion mode at a mass range of 300-1800 m/z. The two most intensely charged peptides above 20 counts were selected for MS/MS at a dynamic exclusion of 30 sec 44 (link) . Data were processed by ProteinPilot v2.0 (AB Sciex) and the candidate proteins were identified. Protein identification was based on a threshold of protein score >1.3, a confidence limit of 95%, a false discovery rate of 5%, and an additional iTRAQ ratios >1.3. For quantitation, at least two unique peptides with 95% confidence and a P-value <0.05 were required. The bioinformatic analysis of gene ontology (GO) was performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID).
3
Isoelectric Focusing and Mass Spectrometry for Peptide Identification
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Labeled peptides were fractionated and puri ed by immobilized-pH-gradient isoelectric focusing (IPG-IEF), as previously described [24] . Puri ed peptide fractions were reconstituted in solvent A. The peptides were electrosprayed using a nanoelectrospray ionization source at an ion spray voltage of 2300 eV and analyzed by a NanoLC-ESI-Triple TOF 5600 system (AB Sciex). The mass spectrometer was set in the positive ion mode at a mass range of 300-1800 m/z. The two most intensely charged peptides above 20 counts were selected for MS/MS at a dynamic exclusion of 30 sec [24] . Data were processed by ProteinPilot v2.0 (AB Sciex) and the candidate proteins were identi ed. Protein identi cation was based on a threshold of protein score >1.3, a con dence limit of 95%, a false discovery rate of 5%, and an additional iTRAQ ratios >1.3. For quantitation, at least two unique peptides with 95% con dence and a Pvalue <0.05 were required. The bioinformatic analysis of gene ontology (GO) was performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID).
4
Peptide Separation and Identification
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Labeled peptides were fractionated and purified by immobilized-pH-gradient isoelectric focusing, as previously described [23, 24] . Purified peptide fractions were reconstituted in solvent A (water/ACN [98:2 v/v] with 0.1% formic acid) and separated using a C18-PepMap column (Thermo Fisher Scientific, Beijing, China) with a solvent gradient of 2-100% Buffer B (0.1% formic acid and 98% acetonitrile) in Buffer A at a flow rate of 0.3 µl/min. The peptides were electrosprayed using a nanoelectrospray ionization source at an ion spray voltage of 2300 eV and analyzed by a NanoLC-ESI-Triple TOF 5600 system (AB Sciex, Framingham, MA). The mass spectrometer was set to positive ion mode at a mass range of 300-1800 m/z. The two most intensely charged peptides above 20 counts were selected for MS/MS at a dynamic exclusion of 30 s [25] . Data were processed using ProteinPilot v2.0 (AB Sciex) and compared with the International Protein Index Human database v3.77. Cysteine modified by methane thiosulfate was specified as a fixed modification. Protein identification was based on a threshold protein score of >1.3. For quantitation, at least two unique peptides with 95% confidence and a P-value less than 0.05 were required.
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