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Nanoparticle-Aided Nanoreactor for Nanoproteomics.

ABSTRACT: Large-scale bottom-up proteomics of few even single cells is crucial for a better understanding of the roles played by cell-to-cell heterogeneity in disease and development. Novel proteomic methodologies with extremely high sensitivity are required for few even single-cell proteomics. Sample processing with high recovery and no contaminants is one key step. Here we developed a nanoparticle-aided nanoreactor for nanoproteomics (Nano3) technique for processing low-nanograms of mammalian cell proteins for proteome profiling. The Nano3 technique employed nanoparticles packed in a capillary channel to form a nanoreactor (≤30 nL) for concentrating, cleaning, and digesting proteins originally in a lysis buffer containing sodium dodecyl sulfate (SDS), followed by nanoRPLC-MS/MS analysis. The Nano3 method identified a 40-times higher number of proteins based on MS/MS from 2-ng mouse brain protein samples compared to the SP3 (single-pot solid-phase-enhanced sample preparation) method, which performed the sample processing using the nanoparticles in a 10 μL solution in an Eppendorf tube. The data indicates a drastically higher sample recovery of the Nano3 compared to the SP3 method for processing mass-limited proteome samples. In this pilot study, the Nano3 method was further applied in processing 10-1000 HeLa cells for bottom-up proteomics, producing 441 ± 263 (n = 4) (MS/MS) and 983 ± 292 (n = 4) [match between runs (MBR)+MS/MS] protein identifications from only 10 HeLa cells using a Q-Exactive HF mass spectrometer. The preliminary results render the Nano3 method a useful approach for processing few mammalian cells for proteome profiling.

SUBMITTER: Yang Z

PROVIDER: S-EPMC9563093 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Nanoparticle-Aided Nanoreactor for Nanoproteomics.

Yang Zhichang Z Zhang Zhaoran Z Chen Daoyang D Xu Tian T Wang Yuan Y Sun Liangliang L

Analytical chemistry 20210723 30

Large-scale bottom-up proteomics of few even single cells is crucial for a better understanding of the roles played by cell-to-cell heterogeneity in disease and development. Novel proteomic methodologies with extremely high sensitivity are required for few even single-cell proteomics. Sample processing with high recovery and no contaminants is one key step. Here we developed a nanoparticle-aided nanoreactor for nanoproteomics (Nano3) technique for processing low-nanograms of mammalian cell prote ...[more]

PMID: 34297524

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Project description:We developed an integrated proteomics approach using a chemically functionalized gold nanoparticle (AuNP) as a novel probe for affinity purification to analyze a large protein complex in vivo. We then applied this approach to globally map the transcriptional activation complex of the estrogen response element (ERE). This approach was designated as quantitative nanoproteomics for protein complexes (QNanoPX). In this approach, the positive AuNP-ERE probes were functionalized with polyethylene glycol (PEG), and the consensus sequence of ERE and negative AuNP-PEG probes were functionalized with PEG without the ERE via a thiolated self-assembly monolayer technique. The AuNP-ERE probe had substantially low nonspecific binding and high solubility, which resulted in a 20-fold enrichment of the factor compared with gel beads. In addition, the surface-only binding allows the probe to capture a large protein complex without any restrictions due to pore size. The affinity purification method was combined with MS-based quantitative proteomics and statistical methods to reveal the components of the ERE complex in MCF-7 cells and to identify those components within the complex that were altered by the presence of 17beta-estradiol (E2). Results indicated that a majority of proteins pulled down by the positive probe exhibited significant binding, and approximately one-half of the proteins, including estrogen receptor alpha (ERalpha), were slightly but significantly affected by a 24-h treatment with E2. Based on a combination of bioinformatics and pathway analysis, most of the affected proteins, however, appeared to be related to the transcriptional regulation of not only ERalpha but also c-Myc. Further confirmation indicated that E2 enhanced the ERE binding of c-Myc by 14-fold, indicating that c-Myc may play a major role, along with ERalpha, in E2-mediated transcription. Taken together, our results demonstrated a successful QNanoPX approach toward new pathway discovery and further revealed the importance of cross-interactions among transcription factors.

Dataset Information

Nanoparticle-Aided Nanoreactor for Nanoproteomics.

Publications

Nanoparticle-Aided Nanoreactor for Nanoproteomics.

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