Nanoparticle-Aided Nanoreactor for Nanoproteomics

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 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 441 ± 263 (n = 4) (MS/MS) and 983 ± 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 proteome profiling.