Thus, it is necessary to analyze the entire set of produced proteins [8]. Proteome analysis of M. loti in mid-growth phase has been reported [9], but it has not been performed for the symbiotic phase. Proteome analyses of other rhizobia, such as B. japonicum[10–14], and S. meliloti[15–20], have been previously reported. They employed 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE)-based analysis combined with matrix-assisted laser desorption and ionization time-of-flight mass spectrometry

(MALDI-MS), but time-consuming steps, such as gel spot isolation and individual measurement, are necessary in this method. In addition, previous 2D-PAGE-based analyses have only identified up to 500 proteins [13]. Another report employed liquid chromatography-tandem Atezolizumab mass spectrometry (LC-MS/MS)-based technology combined with prefractionation, such as multidimensional chromatography VX-809 research buy [21] or gel-based separation [22], but these prefractionation steps decreased throughput. Furthermore, all of them included a complicated isolation step of the bacteroid (a symbiotic form of rhizobia) from the nodule, and the step required a large amount of biological samples, such as 1–5 g nodules collected from approximately 40 plants [23]. Detection of small amount of proteins present in complex biological samples remains difficult

and requires a combination of prefractionation steps. To solve the problems, we used a nanoLC-MS/MS system equipped AZD9291 chemical structure with a long monolithic silica capillary column (200 cm long, 0.1 mm ID). Monolithic silica materials offer high separation efficiency in long column formats because of their high permeability [24], and they have been successfully applied to separate tryptic fragments in highly complex samples with a shallow gradient.

As this high-resolution system does not require any additional prefractionation prior to the separation and detection step by LC-MS/MS, this approach can simplify the workflow of shotgun proteomics and minimize the sample amount, as well as total analysis time [25]. Using this system, we have successfully performed proteome analysis of Candida albicans[26] and Clostridium cellulovorans[27]. Here, we report the first comparative proteome analysis of M. loti under the free-living and symbiotic conditions by using our system. Our data should accelerate functional and comprehensive studies focused on molecular mechanisms of L. japonicus – M. loti symbiosis. Results and discussion Identification of proteins extracted from free-living and symbiotic M. loti The tryptic digests were injected to a LC-MS/MS system equipped with a long monolithic silica capillary column; 1,658 proteins were successfully identified by efficient separation (Additional file 1).