Nanoparticle-aided nanoreactor for large-scale proteomics of few mammalian cells

ABSTRACT: Large-scale bottom-up proteomics of few even single cells is crucial for a better understanding of the roles played by cell-to-cell heterogeneity in disease and development. Novel proteomic methodologies with extremely high sensitivity are required for few even single-cell proteomics. Sample processing with high recovery and no contaminants is one key step. Here we developed a nanoparticle-aided nanoreactor for nanoproteomics (Nano3) technique for processing low-nanograms of mammalian cell proteins for large-scale proteome profiling. The Nano3 technique employed nanoparticles packed in a capillary channel to form a nanoreactor (≤30 nL) for concentrating, cleaning, and digesting proteins originally in a lysis buffer containing sodium dodecyl sulfate (SDS), followed by nanoRPLC-MS/MS analysis. The Nano3 method identified a 40-times higher number of proteins based on MS/MS from 2-ng mouse brain protein samples compared to the SP3 (Single-Pot Solid-Phase-enhanced Sample Preparation) method, which performed the sample processing using the nanoparticles in a 10-µL solution in an Eppendorf tube. The data indicates a drastically higher sample recovery of the Nano3 compared to the SP3 method for processing mass-limited proteome samples. In this pilot study, the Nano3 method was further applied in processing 10-1000 HeLa cells for bottom-up proteomics, producing 444 ± 263 (n=4) (MS/MS) and 987 ± 292 (n=4) [match between runs (MBR)+MS/MS] protein identifications from only 10 HeLa cells using a Q-Exactive HF mass spectrometer. The preliminary results render the Nano3 method a useful approach for processing few mammalian cells for large-scale proteome profiling.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD027135 | Pride | 2022-07-11

REPOSITORIES: Pride