Single cell proteomics is making strides in the field of protein mass spectrometry. Newly customised as well as automated sample preparation techniques are being used to capture and process single cells for proteomic analysis. In addition, the enhanced sensitivity and speed of new-generation mass spectrometers is making way for novel paradigms in acquisition and analysis of samples at single cell resolution. The ability to capture molecular signatures at sub-nanogram levels has been shown to be of enormous significance in studying cell differentiation, cell perturbations and systems biology, just to name a few. However, working at such low concentrations poses certain challenges in terms of establishing and optimising methods that balance the trade-off of throughput and capturing the proteome besides the intricacies of single cell sample preparation.
Using low-/ultra-low nano flow rates with single cell equivalent concentrations from commercially available HeLa digests, we identified about 350 proteins/ ~800 peptides consistently by MS2 at single cell equivalent concentrations on a Thermo ScientificTM Orbitrap ExplorisTM 480 acquired using FAIMS dual CV data dependant acquisition method. As expected, using match between runs (MBR) between individual single cell equivalent acquisitions marginally increased the number of identifications to ~400 proteins/ ~1000 peptides with a majority of them being identified by a single peptide. In contrast, applying MBR using a ten-cell equivalent library (which is the most commonly used approach described in recent literature), we observed a significant increase in the number of identifications to about 1000 proteins. This approach drastically improves the conversion rate of single peptide hits to proteins identified by three or more peptides. Furthermore, we also observed an improvement in the identified proteins in the lower abundance range by an order of magnitude. Going forward, we are currently using these optimised methods to analyse true single cell samples prepared through our in-house microfluidic sample preparation workflow.