den Ridder, Maxime, et al. “A systematic evaluation of yeast sample preparation protocols for spectral identifications, proteome coverage and post-isolation modifications.” Journal of Proteomics 261 (2022): 104576. https://doi.org/10.1016/j.jprot.2022.104576

Abstract

The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols. In particular when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory Saccharomyces cerevisiae. Here, we performed a systematic comparison of sample preparation protocols using a matrix of different conditions commonly applied in whole cell lysate, bottom-up proteomics experiments. The different protocols were evaluated for their overall fraction of identified spectra, proteome and amino acid sequence coverage, GO-term distribution and number of peptide modifications, by employing a combination of database and unrestricted modification search approaches. Ultimately, the best protocols enabled the identification of approximately 65–70% of all acquired fragmentation spectra, where additional de novo sequencing suggests that unidentified spectra were largely of too low spectral quality to provide confident spectrum matches. Generally, a range of peptide modifications could be linked to solvents, additives as well as filter materials. Most importantly, the use of moderate incubation temperatures and times circumvented excessive formation of modification artefacts. The collected protocols and large sets of mass spectrometric raw data provide a resource to evaluate and design new protocols and guide the analysis of (native) peptide modifications.