Ultrasensitive Trace Sample Proteomics Unraveled the Protein Remodeling during Mesenchymal-Amoeboid Transition

Deep mining the proteome of trace biological samples is critical for biomedical applications. However, it remains a challenge due to the loss of analytes caused by current sample preparation procedures. To address this, we recently developed a single-pot and miniaturized in-solution digestion (SMID) method for minute sample handling with three streamlined steps and completed within 3 h. The SMID approach outperformed the traditional workflow in substantially saving time, reducing sample loss, and exhibiting extensive applicability for 10-100 000 cell analysis. This user-friendly and high-sensitivity strategy enables ∼5300 proteins and 53 000 peptides to be confidently identified within 1 h of mass spectrometry (MS) time from a small amount of 1000 HeLa cells. In addition, we accurately and robustly detected proteomes in 10 mouse oocytes with excellent reproducibility. We further adopted SMID for the proteome analysis in cell migration under confinement, which induced cells to undergo a mesenchymal-amoeboid transition (MAT). During the MAT, a systematic quantitative proteome map of 1000 HeLa cells was constructed with seven expression profile clusters, which illustrated the application of SMID and provided a fundamental resource to investigate the mechanism of MAT.