One of the most important steps in quantitative shotgun proteomics analysis is fractionation, at either the protein or peptide level. SDS-PAGE fractionation of proteins, followed by in-gel digestion of proteins is a robust approach that has found widespread application in the field, including in our laboratory. In this study, we set out to determine how an orthogonal fractionation approach compared to our standard workflow in terms of numbers of proteins identified, time involved, ease of use, and applicability to a range of different biological sample types.
Using sample material available from a variety of other projects in our laboratory, we have performed quantitative shotgun proteomics analysis of proteins extracted from a range of biological materials including E. coli, yeast, rice leaf, eucalyptus leaf, human skin and oyster gills. Samples were first analysed using our standard workflow of three replicates of SDS-PAGE fractionation of extracted proteins, followed by in-gel digestion of 16 equal fractions. Samples were then analysed using an alternative workflow involving three replicates of in solution digests of extracted protein using filter assisted sample preparation (FASP), followed by fractionation of peptides into 16 fractions based on hydrophobicity using a reversed-phase spin column. In both cases, peptides were then separated and identified using nanoflow reversed-phase liquid chromatography – tandem mass spectrometry.
Preliminary experiments from analysis of E. coli lysates indicate that the reversed-phase spin column workflow seems to generate more reproducibly identified proteins from the same amount of starting material, and is comparable to the standard workflow in terms of both time involved and ease of use. We will present results from analysis of all the different biological materials involved, including bioinformatic characterisation of the properties of peptides and proteins found to be strongly enriched in one workflow or the other.