Anoliter-scale sample processing and mass spectrometry acquisition method for single cell proteomics

A method of performing proteomic analysis comprising the steps of:

• placing a single cell sample into each of a plurality of nanowells disposed within a chip, the chip also defining a booster well having a volume greater than any single nanowell, the booster well containing a booster sample of known peptides,
• lysing the cell located in the nanowell;
• extracting proteins from the cell to form a nanosample;
• reducing the disulfide groups in the nanosample;
• alkylating the sulfhydryl groups in the nanosample:
• performing a digestion on the nanosample;
• performing tandem mass tag (TMT) labeling on the nanosample;
• acidifying the nanosample;
• collecting nanosamples from at least two nanowells and combining these nanosamples with the booster sample to form a combined booster sample;
• and
• washing the nanowells with a wash solution and adding the used wash solution to the combined booster sample.

A method of performing proteomic analysis comprising the steps of:

• placing a single cell sample into a nanowell disposed within a chip, the nanowell configured to have a diameter <2 mm and hold liquid volume <1 μL;
• placing a peptide mixture into a booster well, the booster well configured to have a diameter >1 mm and hold liquid volume >1 μL;
• lysing the cell sample;
• extracting proteins from the cell sample;
• digesting the proteins into peptides;
• labeling the peptides and the peptide mixture using tandem mass tag (TMT) labels;
• combining the labeled peptides from the nanowells and the booster wells into the booster well to form a mixed sample;
• separating the mixed sample;
• and
• acquiring data from the separated sample using a mass spectrometer.