Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Proteomic analysis of  low numbers of Hela cells using a microfluidic device (#933)

Peng Xue 1 2 , Xiaobao cao 3 , Ludovic gillet 1 , Sofia Theodorou 1 , Federico Uilana 1 , Chen Li 1 4 , Andrew deMello 3 , Rudolf Aebersold 1
  1. Institute of Molecular Systems Biology, ETH, Zurich, zurcih, Switzerland
  2. Institute of Biophysics , Chinese Academy of Sciences, Beijing, China
  3. Department of Chemistry and Applied Biosciences , ETH, Zurich, zurcih, Switzerland
  4. Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia

Single-cell analysis enables the characterization of highly heterogeneous cellular heterogeneity and the complexity of cellular interactions by the collection of genomic, transcriptomic, proteomics and metabolomics information. While genomic and transcriptomic single-cell based approaches are well-established, the proteomic profiling of single-cells is however still debuting as a novel research area. Capturing proteomic information from individual cells is technically challenging in terms of sample handling and preparation, as it is limited by the sensitivity of the mass spectrometry acquisition level and data analysis. Here we present a novel PDMS(Polydimethylsiloxane) microfluidic device for the proteomic analysis of small cell populations. The developed device enables the capturing of few cells ranging from 1 to 200 cells in a multiplexed way to allow paralleled sample preparations. Within the device, cells are subsequently lysed, and the released proteins are then reduced, alkylated and finally digested. All these steps can be performed in a reduced nanoscale volume of a total of 30 nl. The minimum surface of the device dramatically improves the recovery of the peptides during the sample processing due to the peptide nonspecific adsorption surface binding. The digestion efficiency in the chip demonstrated to be equal or even better than the in-solution bulk (1mL) or low volume (10μL) digestion in the tubes. Additionally, we optimized the data-independent acquisition (DIA) sensitivity by increasing the ion injection time to 246ms and setting the resolution to 120,000 at the expense of the mass range covered. With these settings, we were able to reproducibly measure ~500 proteins from 250 pg of peptides (~1 HeLa cell). In conclusion, by combining this microfluidics device to a sensitive and robust quantitative technique based on a data-independent acquisition mass spectrometry method, we have measured reproducibly 2000 protein groups based on 10,000 peptides extracted from a total of 50 Hela cells consistently.