Mass spectrometry acquisition methods for subcellular spatial proteomics: a direct comparison of resolution and reproducibility

Compartmentalisation of eukaryotic cells into organelles allows separate biochemical processes to occur simultaneously. The subcellular localisation of a protein influences its function by defining both the surrounding physical conditions and the biomolecules available for interaction. Movement of proteins between organelles is essential for normal cellular activity, allowing multiple roles to be fulfilled in different environments, and mislocalisation is linked with an increasingly wide range of diseases. The optimisation of techniques for profiling the spatial proteome is imperative, to gain new insight into both normal cellular function and responses to perturbation, especially in cases where overall protein abundance stays constant but localisation changes.
The LOPIT-DC protocol is able to map the subcellular localisation of thousands of proteins in a single experiment by utilising differential ultracentrifugation fractionation, before isotope tagging, DDA mass spectrometry and correlation profiling based analysis. Within the same experimental pipeline, we compare the established DDA-MS based LOPIT-DC method to a DIA-MS approach, for a direct comparison of resolution and reproducibility.
Initial analysis of the resolution suggests the DIA workflow can identify significantly more proteins, with the DDA workflow generally showing tighter organelle marker clustering after PCA analysis. Both methods are effective at distinguishing proteins from nuclear, cytosolic, and mitochondrial compartments, as well as key secretory pathway organelles including the Golgi apparatus and endoplasmic reticulum. The DIA workflow reduces the amount of mass spectrometry instrument time required, since fewer fractions are run. In terms of sample preparation, a lower number of initial cells can be used with the DIA workflow as the amount of protein mass required in each fraction for TMT labelling protocols is no longer a limiting factor.