One of the main goals of global proteomic profiling studies is to achieve sufficient depth to identify and/or quantify all the proteins expressed in a cell or tissue type. LC-MS/MS analysis of tryptic digests from whole cell lysates often shows better coverage for high abundant proteins compared to low abundant proteins. Pre-fractionation methods using chromatography have been widely used to achieve greater depth in proteomics. However, these strategies often show greater coverage for most cytosolic proteins that are more abundant than nuclear proteins including transcription factors that are relatively less abundant. We reasoned that coupling a simple ultracentrifuge independent subcellular fractionation strategy with bRPLC based fractionation will provide better coverage of both cytosolic and nuclear proteins.
A simple method utilizing osmotic lysis and centrifugation was performed on human melanoma cell lines in order to separate them into nuclear and cytoplasmic fractions. These, along with whole cell lysates of the same cell lines were digested using trypsin and fractioned by bRPLC. 10% of each fraction was used for global proteome profiling and the remaining 90% was used for TiO2 based phosphopeptide enrichment. All the samples were subjected to LC-MS/MS analysis on Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer. MS/MS search was carried out using SEQUEST search algorithm in Proteome Discoverer software platform.
Coupling subcellular separation and bRPLC fractionation provided better proteome and phosphoproteome coverage for both cytosolic and nuclear proteins. The strategy also enabled identification of more peptides per protein increasing the confidence in these identifications. Better coverage was observed for low abundant proteins including transcription factors that are often underrepresented in global proteomic datasets. This study demonstrates a relatively easy strategy that can be employed for achieving better proteome and phosphoproteome coverage in global proteome profiling studies.