organelle: vacuoles of C. merolae synchronized at interphase
Extracted molecule
protein
Extraction protocol
The vacuoles of C. merolae synchronized at interphase were isolated by differential and iodixanol-gradient ultracentrifugation. The supernatant (5 ug of protein) were subjected to SDS-PAGE, cut into 69 slices without staining, and subjected to automatic sample preparation for MALDI-TOF-MS by a robot Xcise (Shimadzu). Alternatively, the vacuolar fraction protein (15.8 ug) was alkylated with 5% (w/v) acrylamide, subjected to SDS-PAGE, and stained with Imperial Protein Stain (Pierce). The gel slices of 58 visible bands were destained and digested as described previously (PMID: 11507753). After the digestion, the peptide fragments were extracted from the gel pieces with 5% (v/v) formic acid in 50% (v/v) CH3CN. Extracts were dried in a vacuum concentrator. The dried peptides were dissolved in 0.1% (v/v) trichloroacetic acid (TFA), desalted using uC18-Zip Tip microcolumns (Millipore) and spotted onto a 384-well plate (KRATOS Analytical). After completely drying the samples, 0.5% (w/v) alpha-cyano-4-hydroxycinnamic acid in 50% (v/v) CH3CN and 0.1% (v/v) TFA were spotted on the peptide spots and allowed to dry.
Label protocol
*
Hybridization protocol
*
Scan protocol
*
Description
Identification of proteins from vacuoles of red algae [Cyanidioschyzon merolae strain 10D (Eukaryota; Rhodophyta; Bangiophyceae; Cyanidiales; Cyanidiaceae)] synchronized at interphase, using MALDI-TOF.
Data processing
The samples were analyzed by peptide mass fingerprinting (PMF) using a mass spectrometer (AXIMA-TOF2; Shimadzu) in the reflectron mode. When samples could not be identified by PMF, several peptide peaks were further analyzed by post source decay (PSD)-MS/MS. Database searches were performed using the software program MASCOT v2.2.01 (Matrix Science) running on a local database built from the FASTA files of 5014 open reading frames in the C. merolae genome database (http://merolae.biol.s.u-tokyo.ac.jp/). The peptide tolerance was set to 0.1-0.2 Da. Cysteine carbamidomethylation (for samples prepared by Xcise) or propionamidation (for manually prepared samples) was included as a fixed modification and methionine oxidation was included as a variable modification. The identification threshold was set to p < 0.05. As a result, 46 proteins were identified.
