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

Abstract

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.

Lamin A/C and Vimentin as a Coordinated Regulator during Amoeboid Migration in Microscale Confined Microenvironments

“…To decipher the key protein changes in cells during A2 cell transition, we developed an ultrasensitive trace sample proteomics based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and clearly unraveled the proteomic map. 23 We found a total of 632 differentially expressed proteins (DEPs) from the confined (Conf.) and nonconfined (Nonconf.)…”

An angled-shape tip-based strategy for highly sensitive proteomic profiling of a low number of cells

“…Further extending the time length, the identification number of protein groups decreased. Previous studies have reported that overnight protein digestion may result in ‘ trans -peptides’, a side reaction during digestion 11,26,27 When analyzing complex protein mixtures, the trans -peptide products from abundant proteins may interfere with the low-abundance protein peaks, resulting in a decreased identification number. Reducing the digestion time can avoid this side reaction for better proteomic profiling.…”

“…The false discovery rate (FDR) of peptides lower than 1% was used as the cut-off value for protein identification. 11,25…”

“…MS-based proteomic approaches have enabled the simultaneous investigation of thousands of proteins from complex biological samples. 9,10 Typically, proteomics analysis requires >10 4 mammalian cells 11 for deep coverage due to protein loss during sample processing, such as cell lysis, digestion and peptide desalting. However, bulk samples are not always available in basic biological research and clinical investigations, particularly for stem cell or tumour heterogeneity studies.…”

Mapping the Protein Kinome: Current Strategy and Future Direction

“…To avoid tube-to-tube transfer and related sample losses, it is critical to perform the multistep in a single pot. Thus, multiple methods have been described to minimize sample losses, such as in-StageTip digestion (iST), filter-aided sample preparation (FASP), and single-pot solid-phase-enhanced sample preparation (SP3) [ 127 , 128 ]. These methods have been used for the preparation of paucicellular samples, even a single cell.…”