Group of Omics Analysis, ToMMo
Sample collection and storage
To collect plasma samples, we got the informed consent of more than 500 healthy volunteers living in both Miyagi and Iwate prefectures. Those blood samples were collected in vacutainer tubes containing previously EDTA-2Na. Acquired plasma were divided into 6 aliquots in 1.0 ml screw tube. These samples were administered by ToMMo Omics ID, and stored at -80oC until analysis.
nanoLC-MS/MS system and protein database searching for proteomics
Each proteomic sample was injected into nanoLC system, and eluted with a 180 min gradient of solvent B (0.1% formic acid in acetonitrile, v/v) in solvent A (0.1% formic acid in water, v/v) at a flow rate of 300 nl/min. Those were then ionized and analyzed by a Q-FT-IT/MS, FT-IT/MS, or Q-FT/MS using a nano-spray source. High-resolution full scan MS spectra (from m/z 400-2000) were acquired in the Orbitrap, following by MS/MS fragmentation.
The MS raw data were analyzed by sequence alignment using variable, and static modifications by Mascot and Sequest algorithms. Protein database utilized Swiss-Prot, which is considered each peptide sequences in Trypsin. All peptide identified with peptide score of Mascot were manually examined using rules described previously (Chen Y. et al., J. Proteome Res. 4 (2005) 998-1005).