van Pijkeren, A. | Search bioRxiv

bioRxiv2022

Proteome coverage after simultaneous proteo-metabolome liquid-liquid extraction

Proteo-metabolomics is essential in systems biology and simultaneous proteo-metabolome extraction by liquid-liquid extraction (SPM-LLE) allows extraction of the metabolome and proteome from the same sample. Since the proteome is present as a pellet in SPM-LLE it must be solubilized for quantitative proteomics. Solubilization and proteome extraction is a critical factor in the information that can be obtained at the proteome level. In this study, we investigated the performance of two surfactants (sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS)) and urea with respect to proteome coverage and extraction efficiency of an interphase proteome pellet generated by methanol-chloroform based SPM-LLE. We also investigated the extent to which the performance differs when the proteome is extracted from the interphase pellet or by direct cell lysis. Our study reveals that the proteome coverages between the two surfactants and urea for the SPM-LLE interphase pellet were very similar, but the extraction efficiencies differed significantly. While SDS led to enrichment of basic proteins, which were mainly ribosomal and ribonuclear proteins, urea was the most efficient extraction agent for simultaneous proteo-metabolome analysis. The results of our study also show that the performance of surfactants (SDC, SDS) for quantitative proteomics is better when the proteome was extracted by direct cell lysis and not from an interphase pellet. In contrast, the performance of urea for quantitative proteomics was significantly better when the proteome was extracted from an interphase pellet and by direct cell lysis.

systems biology↗

bioRxiv2020

Natural isotope correction improves analysis of protein modification dynamics

Stable isotope labelling in combination with high resolution mass spectrometry approaches are increasingly used to analyse both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example.

bioinformatics↗