Analysing the measurable proteome in complex tissue samples using mass spectrometry (MS) can be time-consuming with traditional methods. Pressure cycle technology (PCT) significantly reduces sample preparation time, and increases robustness and consistency. When coupled with SWATH-MS all detectable peptides in a sample can be quantified in a one single run. Our aim was to test the capabilities of PCT-SWATH-MS so see if the sensitivity could be compromised due to high acquisition speed. We tested the methods capability to quantify differential protein distribution in subregions within an body organ.
We chose to use five different brain regions, motor- and somatosensory cortex, hippocampus, amygdala and thalamus. Samples were collected in triplicate from each region with a biopsy punch from three different 24 week old Sprague Dawley rats. Samples were prepared in three different PCT instruments (Barocyclers) for homogenisation and tryptic digestion. The digests were run in duplicates on a Sciex TripleTOF 6600. The resultant SWATH-MS data were analysed and quantified using an internal rat brain spectral reference library (SRL) that was generated on the same type of instrument and HPLC gradient using standard shotgun methods. Data visualisation and sample QC was performed with Skyline, MarkerView, VennDIS and Panther.
The data showed that the fast 6 hour sample preparation from PCT did not comprise peptide concentration (2.5 µg peptides per µL) and consistency of digestion efficiency (80%). The SWATH-MS data identified about 3,000 proteins and 11,000 peptides in each sample, suggesting good depth. In MarkerView three protein clusters were observed: 1) motor- and somatosensory cortex, 2) amygdala and hippocampus, 3) thalamus. Each region had between 30 to 60 unique proteins visualised with VennDIS, with the somatosensory cortex having the highest amount of 62 unique proteins out of 2,856 proteins (2%). The unique proteins found in each brain region did not have common gene ontology pathways. Compared to traditional sample preparation and shotgun MS methods, PCT-SWATH-MS reduced sample preparation from traditional taking 12-24 hours to 5-6 hours and the label-free quantification reduced the cost significantly. The results show that PCT-SWATH-MS is a high throughput and reliable method for detection and label-free quantification of small differences in protein expression between different brain regions.