Single-cell proteomics (SCP) is enabling researchers to understand cancer progression, immune response, developmental pathways, and more all at the cellular level. Historically, SCP has represented a considerable challenge for researchers due to low amounts of proteins within a single cell and the limited sensitivity of mass spectrometry instruments. These hurdles, along with others such as ion signal suppression, sufficiently efficient data acquisition methods, and variability in sample preparation can obfuscate data and limit discoveries.
In response, researchers from the Broad Institute of MIT and Harvard have developed the proteoCHIP EVO 96, which is poised to bridge the gap. The proteoCHIP EVO 96 workflow, recently published in Nature Communications, relies on several cutting-edge technologies to automate key steps in SCP sample preparation and analysis with hopes of improving reproducibility, reducing variability, and increasing throughput.
ProteoCHIP EVO 96
The proteoCHIP EVO 96 itself is a micromachined PTFE (polytetrafluoroethylene) chip specifically designed to combat protein loss. Traditional workflows involve transferring samples between containers multiple times. These transfers can result in losses as proteins adsorb to the walls of each container used.
The proteoCHIP ensures that proteins remain within the same nanowells throughout the entire preparation process by employing a 'one-pot' sample preparation method. All sample preparation steps, including cell lysis, protein digestion, and peptide cleanup, are conducted in the same nanowell.
CellenONE
The one-pot method is enabled by cellenONE, an automated dispensing robot responsible for populating the 96 nanowells of the proteoCHIP EVO 96. It delivers individual cells, dispenses reagents, such as detergents for cell lysis and proteases for protein digestion, and ensures consistent hydration and mixing throughout.
After the proteins are digested in the proteoCHIP EVO 96, the chip is inverted for direct transfer to disposable trap columns for liquid chromatography via the Evosep One. The group found that introducing just one manual sample transfer step at this stage of the workflow (manually pipetting samples from nanowells to disposable trap columns) reduces total protein identifications by up to 29%.
Bruker timsTOF Ultra
The peptides are chromatographically separated and directly analyzed by the timsTOF Ultra mass spectrometer, equipped with Trapped Ion Mobility Spectrometry (TIMS) technology. TIMS separates ions based on their shape followed by size selection in the quadrupole before they reach the mass analyzer, which increases the overall ion utilization and enhances the detection of low-abundance proteins.
In addition the team employs Data-Independent Acquisition (DIA). While Data-Dependent Acquisition (DDA), can miss lower-abundance peptides in some samples due to the stochastic selection of precursor ions, DIA ensures that all ions within a predefined mass range are fragmented and analyzed in each sample.