Global proteome discovery using an online three-dimensional LC-MS/MS.

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.

Desorption/ionization on silicon time-of-flight/time-of-flight mass spectrometry.

Desorption/ionization on silicon (DIOS) tandem time-of-flight (TOF/TOF) mass spectrometry (MS) provides high accuracy and significant fragmentation information, particularly in the characterization of biomolecules. DIOS TOF/TOF offers a high-throughput surface-based ionization platform as well as complete fragmentation through high collision energies. The absence of matrix interference in DIOS allows for the MS and MS/MS analysis of small molecules well below m/z 300. In addition, sample preparation is minimal, and the DIOS chips can be stored and reanalyzed for fragmentation information or accurate mass measurements. The combined benefits of robustness, minimal sample preparation, good sensitivity, high throughput, and sequencing capability make DIOS TOF/TOF a powerful tool for small molecule characterization and protein identification.