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A simple and flexible strategy for single-cell proteomic analysis based on protein immobilization and digestion tube reactor

ABSTRACT: Mass spectrometry-based single-cell proteomics (SCP) has witnessed rapid development over the past 10 years. However, the current preprocessing methodologies mostly require either proficient operation skills or reliance on costly consumables and advanced instruments, which hinders the widespread application of deep single-cell proteomics. To address this challenge, we have developed a protein immobilization-based microliter reactor (PIBMR), to achieve streamlined and low-cost protein preprocessing for multiple single-cell samples within one hour using common laboratory consumables, and the processed samples are directly compatible with commercial liquid chromatography-mass spectrometry (LC-MS/MS) system. The MiSPIDR workflow integrates the protein processing steps prior to LC-MS/MS analysis into two steps, which requires only microliter-level precision in liquid handling. Additionally, the workflow expands the range of lysis reagents available by employing a covalent immobilization strategy, achieving low-loss protein purification. Using the MiSPIDR workflow in DIA mode, 3732-4651, 2290-3808, and 3423-4469 protein groups were identified in single A549 cells (n=16), HeLa cells (n=16), and MCF-7 cells (n=16), respectively. Furthermore, utilizing the MiSPIDR workflow, we investigated the proteomic heterogeneity of cervical cancer cells at different apoptotic stages following paclitaxel treatment at the single-cell level, demonstrating the potential of single-cell proteomics in addressing biological problems.

ORGANISM(S): Homo Sapiens

SUBMITTER: Xiangmin Zhang

PROVIDER: PXD070012 | iProX | Tue Oct 28 00:00:00 GMT 2025

REPOSITORIES: iProX

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Project description:Single-cell proteomic analysis provides valuable insights into cellular heterogeneity allowing the characterization of the cellular microenvironment which is difficult to accomplish in bulk proteomic analysis. Currently, single-cell proteomic studies utilize data-dependent acquisition (DDA) mass spectrometry (MS) coupled with a TMT labelled carrier channel. Due to the extremely imbalanced MS signals among the carrier channel and other TMT reporter ions, the quantification is compromised. Thus, data-independent acquisition (DIA)-MS should be considered as an alternative approach towards single-cell proteomic study since it generates reproducible quantitative data. However, there are limited reports on the optimal workflow for DIA-MS-based single-cell analysis. Herein, we report an optimized DIA workflow for single-cell proteomics using Orbitrap Lumos Tribrid instrument. We utilized a breast cancer cell line (MDA-MB-231) and induced drug resistant polyaneuploid cancer cells (PACCs) to evaluate our established workflow. We found that a short LC gradient was preferable for peptides extracted from single cell level with less than 2 ng sample amount. The total number of co-searching peptide precursors was also critical for protein and peptide identifications at nano- and sub-nano-gram levels. Post-translationally modified peptides could be identified from a nano-gram level of peptides. Using the optimized workflow, up to 1,500 protein groups were identified from a single PACC corresponding to 0.2 ng of peptides. Furthermore, about 200 peptides with phosphorylation, acetylation, and ubiquitination were identified from global DIA analysis of 100 cisplatin resistant PACCs (20 ng). Finally, we used this optimized DIA approach to compare the whole proteome of MDA-MB-231 parental cells and induced PACCs at a single-cell level. We found the single-cell level comparison could reflect real protein expression changes and identify the protein copy number. Our results demonstrate that the optimized DIA pipeline can serve as a reliable quantitative tool for single-cell as well as sub-nano-gram proteomic analysis.

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A simple and flexible strategy for single-cell proteomic analysis based on protein immobilization and digestion tube reactor

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