Ltq ion trap ms

The identification of proteins in the bands of interest observed on SDS-PAGE gels was conducted via liquid chromatography/tandem mass spectrometry (LCMS/MS). The peptides were analyzed using LTQ ion trap MS (Thermo Fisher Scientific, Waltham, MA, USA). In brief, the gel bands were excised from the colloidal Coomassie blue-stained gels and proteolyzed with trypsin (Promega, Madison, WI, USA). The peptides were fractionated by reverse-phase high-performance liquid chromatography with an emitter column size of 10 μm using a 0–60% acetonitrile/0.5% formic acid gradient with a flowrate of 300 nL/min over 30 min. Peptide sequences were identified using Mascot (www.matrixscience.com, accessed on 1 January 2012) or Sequest (https://proteomicsresource.washington.edu/protocols06/sequest.php, accessed on 1 January 2012) software to search the National Center for Biotechnology Information non-redundant database with the acquired fragmentation data. Identified sequences were confirmed by manually inspecting the fragmentation spectra.

Proteins were extracted from the EBCs from the roots, and ∼50 μg of sample was separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and stained with Coomassie blue. Gel strips containing proteins ranging from 21.5 to 66.2 kDa (NifHDK protein sizes) were excised (Figure 1). Proteome analysis was performed as described previously (Kasahara et al., 2012 (link); Bao et al., 2014 (link)). Briefly, the gel lanes were cut into strips of approximately 1 mm, and the gel strips were digested with trypsin. Nano-liquid chromatography (LC)–electrospray ionization–tandem mass spectrometry (MS/MS) analysis of the peptide mixtures was performed with an LTQ ion-trap MS (Thermo Fisher Scientific) coupled with a multidimensional high-performance LC (HPLC) Paradigm MS2 chromatograph (AMR, Inc., Tokyo, Japan) and nanospray electrospray ionization device (Michrom Bioresources, Inc., Auburn, CA, United States). The MS/MS data were searched against the protein database constructed in this study using Mascot program ver. 2.4 (Matrix Science, London, United Kingdom).