Two-dimensional proteome reference map of Herbaspirillum seropedicae proteins

Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that belongs to the beta-subgroup of Proteobacteria. It is found in association with economically important Poacea, such as sorghum, maize, rice [1], and also banana and pineapple [2]. The genome sequence of H. seropedicae has recently been determined by the GENOPAR Consortium and is estimated to encode approximately 50 00 proteins. Reference 2-D maps in which the proteins are identified can help physiological proteomic studies. This study represents the first step in the construction of a reference map for H. seropedicae. 2-D gels of proteins extracted from H. seropedicae strain SMR1 grown under aerobic NH4-(+)sufficient conditions at 30 degrees C in NFbHP-malate medium [3] were used to create a protein reference map. IEF was performed with commercially available IPG-strips (13 cm, pH 3-10; GE Healthcare, Uppsala, Sweden) using an Ettan IPGphor IEF System (GE Healthcare). The second dimension was performed using 12.5% T-and 2.6% C-polyacrylamide gels in the Ruby SE 600 vertical electrophoresis system (GE Healthcare). Gel analysis was performed using the software ImageMaster 2D Platinum (GE Healthcare). After staining with colloidal CBB [41, the spots were excised manually and in-gel-digested overnight at 37 degrees C with sequencing grade, modified trypsin (Promega, Madison, USA) essentially as described by Westermeier and Naven [4]. The samples were desalted using PerfectPure C-18 tips (Eppendorf, Hamburg, Germany) according to the manufacturer's protocol and the peptides were eluted directly onto the MALDI target. Mass spectra were acquired using a MALDI-TOF-MS Autoflex spectrometer (Bruker Daltonics, Bremen, Germany) in a positive ion reflection/delayed extraction mode with an accelerating voltage of 20 W, delay time of 150 ns and acquisition mass range 800-3200 Da. External calibration was performed using a peptide calibration kit in the mass range 800-3200 Da (Bruker Daltonics). For internal calibration, known autolysis peaks from porcine trypsin were used. Database searches were performed using the ProteinProspector v3.2.1 software on a local server and the gene products of H. seropedicae sequence (www.genopar.org). Peak lists were created using the FlexAnalysis 2.0 software (Bruker Daltonics). The sophisticated numerical annotation procedure (SNAP) algorithm was used to detect the monoisotopic peak values, with a quality factor threshold of 30 and 6 as S/N threshold. The