Introduction
Single cell proteomics is a relatively young niche of proteomics compared to single cell genomics. In the recent years, significant progress has been made in sample handling and boosting the sample signal by multiplexing with mass spectrometers. timsTOF SCP is the first commercially introduced mass spectrometer dedicated for single cell proteomics. The modified front-end (orthogonal ion-guiding) of the instrument increase the ion transfer up to five times and keeps ultra-high robustness – the default attribute of the timsTOF platform. Here we demonstrate the performance of the instrument for low sample loads in the range of 250 pg to 1 ng in combination with robust low flow rate delivery from the Evosep system.
Methods
Commercially available K562 tryptic peptide digests (Promega) were loaded on the Evotips according to the recommended protocol provided by the vendor. Peptide amounts from 125 pg to 25.6 ng were used to evaluate the performance of the instrument. The Whisper low flow method – 40 samples per day (SPD) with a gradient time of about 28 minutes was used for separating the eluting peptides on a 15 cm column with 75 µm ID coupled to captive spray ionization source using a 10-micron ID zero-dead volume emitter. Samples were analyzed using a dia-PASEF method tuned for high sensitivity measurements. Raw data were processed in Spectronaut 15.
Preliminary Data
Processing of diaPASEF data was performed with Spectronaut 15 using a spectral library of 5,200 protein groups and 54,000 peptides and a further improved library with 9,300 protein groups and 116,000 peptides, respectively. Using the combination of Whisper 40 SPD and the high sensitivity methods on the timsTOF SCP, we could quantify around 1,500 protein groups from 250 pg sample loads and close to 2,000 protein groups from 500 pg loads. Performing a dilution series experiments, around 4,500 protein groups could be quantified in 6.4 ng peptide loads.