Single-Cell Proteomics Using the One-Step Droplet-in-Oil Digestion Method Reveals Proteins Important for Male Meiotic Progression

Different
from DNA or RNA, proteins cannot be amplified. The performance
of single-cell proteomics is limited by sample loss during sample
preparation. Here, we present a one-step droplet-in-oil digestion
(OSDO) method that involves one-step water-in-oil processing using
cyclohexane or n-heptane, which can reduce the sample
adsorption loss and sample volume change due to evaporation and increase
the sensitivity and stability of single-cell proteomics. The OSDO
is demonstrated to be compatible with tandem mass tag (TMT) labeling
to improve the throughput. The OSDO, followed by data-independent
acquisition (DIA), quantified more than 3700 proteins per cell during
meiotic progression from pachytene to metaphase I, with no correlation
between protein and mRNA levels. Inhibition of VPS34 and DNMT1, two
proteins up-regulated in metaphase I, both affected metaphase I formation.
The OSDO is an easy-to-operate method compatible with both subsequent
labeled and unlabeled quantification to expand the depth, throughput,
and applicability in single-cell proteomics.