期刊
JOURNAL OF PROTEOME RESEARCH
卷 21, 期 8, 页码 1986-1996出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.2c00274
关键词
digital micro fl uidics; Caenorhabditis elegans; PALMS; label-free quanti fi cation; low-input proteomics; microproteomics; nanoproteomics
资金
- Deutsche Forschungsgemeinschaft (DFG) [SFB1182]
- Cluster of Excellence Precision Medicine in Chronic Inflammation (PMI), RTF-V
This study demonstrates a highly sensitive sample preparation workflow using digital microfluidics and high-resolution mass spectrometry for nanoproteomics research.
ABSTRACT: Miniaturization of sample preparation, including omissible manual sample handling steps, is key for reproducible nanoproteomics, as material is often restricted to only hundreds of cells or single model organisms. Here, we demonstrate a highly sensitive digital microfluidics (DMF)-based sample preparation workflow making use of single-pot solid-phase enhanced sample preparation (SP3) in combination with high-field asymmetric-waveform ion mobility spectrometry (FAIMS), and fast and sensitive ion trap detection on an Orbitrap tribrid MS system. Compared to a manual in-tube SP3supported sample preparation, the numbers of identified peptides and proteins were markedly increased, while lower standard deviations between replicates were observed. We repeatedly identified up to 5000 proteins from single nematodes. Moreover, label-free quantification of protein changes in single Caenorhabditis elegans treated with a heat stimulus yielded 45 differentially abundant proteins when compared to the untreated control, highlighting the potential of this technology for low-input proteomics studies. LCMS data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD033143.
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