High-Throughput Approach for Global Proteomic Measurements
Contact: Dick Smith
The proteomics strategy we have developed is a variation of the "shotgun" proteomics approaches. As with shotgun approaches, proteins are first cleaved into peptide fragments (e.g., by a specific proteolytic enzyme such as trypsin) after cell lysis and sample processing, and liquid chromatography (LC) tandem mass spectrometry (MS/MS) proteome analyses are used to identify peptides. However, in our strategy, these multiple analyses need only be performed once for a particular biological system.
The results from the initial LC-MS/MS analyses are used to create a "look-up table" that contains a characteristic accurate mass and LC separation elution time for each peptide; that is, a mass and time tag, which serves as a unique 2D marker for subsequent identifications of that particular peptide. Once this look-up table (referred to as a mass and time tag database) is created, a future sample need only be analyzed by high-resolution capillary LC-Fourier transform ion cyclotron resonance (FTICR) to detect peptides with the same accurate mass and same elution time characteristics as in the mass and time tag database. This approach not only eliminates the need for routine MS/MS, but also provides both greater analytical sensitivity and increased analysis throughput.
Accurate Mass and Time (AMT) Tag Strategy Full Image (jpg 65kb)
The peptide mass and time tags are assigned using conventional software tools (e.g., SEQUEST) and reflect both the calculated accurate mass and the normalized LC elution time (to <~2% uncertainty based on current approaches). Subsequent identification by LC-FTICR accurate mass measurements effectively validates a putative mass and time tag as an "accurate mass and time" (AMT) tag. This AMT tag approach is similar to identifying a protein spot in a 2D-polyacrylamide gel electrophoresis (PAGE) study. Once a protein spot has been identified in a gel for a particular biological system, a spot at the exact same 2D location in a subsequent analysis of the same system can generally be identified with high confidence.
Analogously, once a mass and time tag has been established for a particular biological system, a peptide eluting at the same normalized time and with the same mass in a subsequent analysis of the same system can generally be identified with high confidence. The confidence of the identification will depend strongly on the specificity provided by both the separation and the accuracy of the mass measurements.