Development of Data Independent Acquisition Techniques for the Analysis of Protein Mixtures by Tandem Mass Spectrometry
Egertson, Jarrett David
MetadataShow full item record
Novel algorithms and data acquisition methods designed to improve the ability to identify and quantify proteins in complex biological mixtures using tandem mass spectrometry are presented. An algorithm for de novo correction of mass-to-charge measurements in MS/MS spectra acquired in a shotgun proteomics workflow is described. The correction of m/z measurements makes the interpretation of MS/MS spectra easier and can increase the number of peptides identified in a bottom-up shotgun proteomics experiment. The technique is described as de novo because it can detect systematic mass measurement error in a collection of tandem mass spectra without any input besides the spectra themselves. To improve quantitation of peptides, a multiplexed data independent acquisition (DIA) technique is presented. Data acquired using DIA can be queried for data on virtually any protein. These data can be used to compare the abundances of proteins in multiple samples with high sensitivity (often higher than using MS data). However, DIA techniques have typically had low precursor selectivity compared to popular data dependent acquisition (DDA) techniques resulting in mixed MS/MS spectra that are difficult to interpret and prone to chemical interference. A method for overcoming this limitation of DIA by multiplexing (and computational demultiplexing) is described. DIA and DDA techniques are used to study the response of the budding yeast S. cerevisiae to treatment with rapamycin. The response to rapamycin is of interest because it extends lifespan in a wide range of organisms. Spectral counting for peptide quantitation using DDA data is compared to quantitation using MS/MS fragment ion chromatograms integrated over time with DIA data.
- Genetics