Oral Presentation 28th Annual Lorne Proteomics Symposium 2023

Analysis of cellenONE sorted and prepared single cells in a label-free nano-flow LC-MSMS proteomics approach using a nanoElute 2 and an Evosep One system and a timsTOF SCP (#53)

Christoph Krisp 1 , Dorte B Bekker-Jensen 2 , David Hartlmayr 3 , Anjali Seth 3 , Moritz Heusel 2 , Magnus Huusfeldt 2 , Thorsten Ledertheil 1 , Jean-Francois Greisch 1 , Andreia Almeida 4 , Jarrod Sandow 4 , Guilhem Tourniaire 3 , Nicolai Bache 2 , Markus Lubeck 1 , Gary Kruppa Kruppa 1
  1. Bruker Daltonics GmbH & Co.KG, Bremen, Germany
  2. Evosep, Odense, Denmark
  3. Cellenion, Lyon, France
  4. IonOpticks, Melbourne, Australia

Introduction

Single cell proteomics is a rapidly developing field with the potential to make important contributions to the understanding of cellular heterogeneity. Single cell protein extraction, minimal exposure of samples to surfaces and optimal storage and transfer conditions are crucial for loss-less single cell proteome analyses. Recent enhancements in trapped ion mobility spectrometry (TIMS) coupled to fast and sensitive mass spectrometry established in the timsTOF SCP paired with automated single cell sorting and sample preparation realized with the cellenONE® platform allows for sensitive proteome analyses at the single cell level.

Methods

Single HeLa cells were sorted into the label-free proteoCHIP®, directly lysed, and proteins digested at 50°C with high humidity on deck using the cellenONE platform. The label-free proteoCHIP with tryptic peptides was placed into the nanoElute 2 autosampler, peptides injected onto a 25 cm Aurora C18 column (IonOpticks) and eluted into a timsTOF SCP. cellenONE Sorted and prepared single HeLa cells were loaded onto Evosep Pure tips and analyzed with an Aurora Elite column at 50 °C using the Whisper 40 SPD method and a timsTOF SCP. dia-PASEF® mode was used and analyzed with TIMS-DIA-NN on PaSER® using a library generated from a deeply fractionated human cell line.

Results

Sample pick-up directly from the label-free proteoCHIP was assessed with HeLa lysate digests (Pierce) showing excellent reproducibility at various concentrations. Injections of 250 pg of HeLa peptides on column (1 µL in well) resulted in 15,000 peptides from 2,600 proteins which was matched by 250 pg HeLa peptides injected from a vial (250 pg/µL). We then analyzed single HeLa cells which were directly sorted and prepared in the label-free proteoCHIP and identified in average more than 2000 proteins per single cell with good reproducibility.

Further, we analyzed a HeLa peptide dilution series ranging from 62.5 pg to 32 ng (n = 6) loaded onto Evptips Pure. From the 250 pg load, we identified 12,000 peptides from 2,500 proteins and from the 32 ng load 70,000 peptides from 7,000 proteins with excellent reproducibility. Sorted single HeLa cells prepared in the label-free proteoCHIP were loaded onto Evotips Pure and analyzed in Whisper 40 SPD leading to more than 10,000 peptides and 2,000 proteins identified from a single cell.

Conclusion

Label-free analysis workflow that reproducibly identifys >2000 proteins from single HeLa cells, using the cellenONE platform with the lable-free proteoCHIP, nanoElute 2, Evosep One and the timsTOF SCP.