Protein bands separated by SDS-PAGE were excised and digested with trypsin as described previously [47 (link)]. Peptide identification was performed as described previously [48 (link)]. Digested peptides were applied onto a Magic C18 AQ nano column (0.1 × 150 mm, MICHROM Bioresources, Inc.) in an ADVANCE UHPLC system (MICHROM Bioresources, Inc.) equilibrated with 0.1% formic acid (v/v) in acetonitrile and eluted using a linear gradient of 5–45% (v/v) acetonitrile at a flow rate of 500 nL/min. Mass analysis was performed using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) with Xcalibur software ver. 2.0.7 (Thermo Fisher Scientific). Peptides were identified using a MASCOT MS/MS ion search (http://www.matrixscience.com/home.html ) in error tolerance mode (one amino acid substitution allowed) using the NCBI database. Search parameters were as follows: taxonomy, plants; max missed cleavages, 0; fixed modifications, carbamidomethyl; peptide tolerance, ± 5 ppm; fragment mass tolerance, ± 0.6 Da.
Xcalibur software ver 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Xcalibur software ver. 2.0.7 is a data acquisition and processing software for mass spectrometry instrumentation. It provides a user interface for instrument control, data collection, and data analysis.
Automatically generated - may contain errors
2 protocols using xcalibur software ver 2
1
Peptide Identification via Mass Spectrometry
2
Mass Spectrometry-Based Protein Identification
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Protein bands separated by SDS-PAGE were excised and digested with trypsin as described previously (Kawamura and Uemura 2003) . Peptide identification was carried out as described previously (Takeda et al. 2015) . Digested peptides were applied onto a Magic C18 AQ nano column (0.1×150 mm, MICHROM Bioresources, Inc., CA, USA) in an ADVANCE UHPLC system (MICHROM Bioresources, Inc.) equilibrated with 0.1% formic acid (v/v) in acetonitrile and eluted using a linear gradient of 5-45% (v/v) acetonitrile at a flow rate of 500 nL/min. Mass analysis was performed using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, MA, USA) operating Xcalibur software ver. 2.0.7 (Thermo Fisher Scientific). Peptides were identified using a MASCOT MS/MS ion search (http://www.matrixscience.com/home.html) in error tolerance mode (one amino acid substitution allowed) using the NCBI database. Search parameters were as follows: taxonomy, plants; max missed cleavages, 0; fixed modifications, carbamidomethyl; peptide tolerance, ± 5 ppm; fragment
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