Pick-up Single-Cell Proteomic Analysis for Quantifying up to 3000 Proteins in a Tumor Cell

Abstract

The shotgun proteomic analysis is currently the most promising single-cell protein sequencing technology, however its identification level of ~1000 proteins per cell is still insufficient for practical applications. Here, we develop a pick-up single-cell proteomic analysis (PiSPA) workflow to achieve a deep identification capable of quantifying up to 3000 protein groups in a tumor cell using the label-free quantitative method. The PiSPA workflow is specially established for single-cell samples mainly based on a nanoliter-scale microfluidic liquid handling robot, capable of achieving single-cell capture, pretreatment and injection under the pick-up operation strategy. Using this customized workflow with remarkable improvement in protein identification, 1804-3349, 1778-3049 and 1074-2487 protein groups are quantified in single A549 cells (n = 37), HeLa cells (n = 44) and U2OS cells (n = 27), respectively. Benefiting from the flexible cell picking-up ability, we study tumor cell migration at the single cell proteome level, demonstrating the potential in practical biological research from single-cell insight.

Ultrasensitive Proteomics Depicted an In-depth Landscape for Mouse Embryo

“…Subsequently, diaPASEF, which combines the advantages of DIA mode acquisition and the high efficiency of PASEF, was developed and applied, suitable for proteomic characterization of very limited samples [26,43–46]. Wang et al demonstrated that diaPASEF can dramatically double the protein identification numbers compared with ddaPASEF in single mammal somatic cells [28]. Our data also showed 120% ∼ 135% improvement of protein identification using diaPASEF compared with ddaPASEF, and the higher correlation of diaPASEF proved the data reliability.…”

“…The copyright holder for this preprint this version posted January 6, 2023. ; https://doi.org/10.1101/2023.01.06.523005 doi: bioRxiv preprint 2,000 proteins can be quantified reliably [26][27][28]. There are also several studies that have applied single-cell proteomic techniques to the oocyte maturation.…”

“…Therefore, the large number of embryos consumed has discouraged many researchers and limited the development of related fields. The good news is that, with the progress of mass spectrometry and the innovation of single-cell analysis strategies in recent years, it has become possible to illustrate the proteomic profiling of trace or even single cells based on MS. From one mammalian somatic cell, more than 2,000 proteins can be quantified reliably [26][27][28]. There are also several studies that have applied single-cell proteomic techniques to the oocyte maturation.…”

Recent advances in microfluidics for single-cell functional proteomics

“…The PiSPA platform demonstrated the highest sensitivity with significant improvement in proteome depth over current SCP methods. 198…”

Simultaneous transcriptome and proteome profiling in a single mouse oocyte with a deep single-cell multi-omics approach

“…The mass spectrometry (MS)-based technique coupled with the shotgun strategy provides an effective way to achieve non-targeted and deep single-cell proteome analysis. Recently, some MS-based systems such as SCoPE-MS 19 , Nested Nanowell chip 20,21 , Evosep 22 , and PiSPA 23 could achieve much deeper proteomic analysis with over 1000 protein groups identified in single cells, and even reaching up to 3000 protein groups using the PiSPA platform. These progress demonstrated a significant breakthrough in single-cell protein identification depth, although they have not reached a comparable level to transcriptome sequencing.…”