A combined database related and de novo MS-identification of yeast mannose-1-phosphate guanyltransferase PSA1 interaction partners at different phases of batch cultivation

Abstract

Mass spectrometry-based proteomic research has become one of the main methods in protein–protein interaction research. Several high throughput studies have established an interaction landscape of exponentially growing Baker's yeast culture. However, many of the protein–protein interactions are likely to change in different environmental conditions. In order to examine the dynamic nature of the protein interactions we isolated the protein complexes of mannose-1-phosphate guanyltransferase PSA1 from Saccharomyces cerevisiae at four different time points during batch cultivation. We used the tandem affinity purification (TAP)-method to purify the complexes and subjected the tryptic peptides to LC–MS/MS. The resulting peak lists were analyzed with two different methods: the database related protein identification program X!Tandem and the de novo sequencing program Lutefisk. We observed significant changes in the interactome of PSA1 during the batch cultivation and identified altogether 74 proteins interacting with PSA1 of which only six were found to interact during all time points. All the other proteins showed a more dynamic nature of binding activity. In this study we also demonstrate the benefit of using both database related and de novo methods in the protein interaction research to enhance both the quality and the quantity of observations.