Applicability of Multidimensional Fractionation to Affinity Purification Mass Spectrometry Samples and Protein Phosphatase 4 Substrate Identification
Year: 2012
Type: dissertation
Abstract: Affinity-purification coupled to mass spectrometry (AP-MS) is gaining widespread use for the identification of protein-protein interactions. It is unclear however, whether typical AP sample complexity is limiting for the identification of all protein components using standard one-dimensional LC-MS/MS. Multidimensional sample separation is a useful for reducing sample complexity prior to MS analysis, and increases peptide and protein coverage of complex samples, yet the applicability of this approach to AP-MS samples remains unknown. Here I present work to show that multidimensional separation of AP-MS samples is not a cost-effective method for identifying increased peptide or protein coverage in these sample types. As such this approach was not adapted for the identification of putative Phosphoprotein Phosphatase 4 (PP4c) substrates. Instead, affinity purification coupled to one-dimensional LC-MS/MS was used to identify putative PP4c substrates, and semi-quantitative methods applied to identify possible PP4c targeted phosphosites in PP2A subfamily phosphatase inhibited (okadaic acid treated) cells.
Source: TSpace (University of Toronto)
Author: Wade H. Dunham
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2758417532