Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics.

We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.

Easy and Accessible Workflow for Label-Free Single-Cell Proteomics.

Single-cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often requires custom instrumentation, consumables, and reagents for sample processing and separations, which has limited the accessibility of SCP to a small number of specialized laboratories. Commercial platforms have become available for SCP cell isolation and sample preparation, but the high cost of these platforms and the technical expertise required for their operation place them out of reach of many interested laboratories. Here, we assessed the new HP D100 Single Cell Dispenser for label-free SCP. The low-cost instrument proved highly accurate and reproducible for dispensing reagents in the range from 200 nL to 2 µL. We used the HP D100 to isolate and prepare single cells for SCP within 384-well PCR plates. When the well plates were immediately centrifuged following cell dispensing and again after reagent dispensing, we found that ∼97% of wells that were identified in the instrument software as containing a single cell indeed provided the proteome coverage expected of a single cell. This commercial dispenser combined with one-step sample processing provides a very rapid and easy-to-use workflow for SCP with no reduction in proteome coverage relative to a nanowell-based workflow, and the commercial well plates also facilitate autosampling with unmodified instrumentation. Single-cell samples were analyzed using home-packed 30 µm i.d. nanoLC columns as well as commercially available 50 µm i.d. columns. The commercial columns resulted in ∼35% fewer identified proteins. However, combined with the well plate-based preparation platform, the presented workflow provides a fully commercial and relatively low-cost alternative for SCP sample preparation and separation, which should greatly broaden the accessibility of SCP to other laboratories.