期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 -, 期 -, 页码 -出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202301969
关键词
Ion Mobility; Mass Spectrometry; Microfluidics; Proteoforms; Proteomics
Researchers have developed a sample preparation method based on protein aggregation and acidic elution, allowing for sensitive protein identification analysis of single nematodes on a digital microfluidics device. Compared to in-tube sample preparation, this method increases proteoform identifications by 46%. Label-free quantification also reveals changes in proteoform abundance that cannot be distinguished by bottom-up proteomics. This workflow facilitates proteoform-focused analysis on limited availability samples.
While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up proteomics workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40 % formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analyses of single Caenorhabditis elegans nematodes, thereby increasing the number of proteoform identifications compared to in-tube sample preparation by 46 %. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in the abundance of proteoforms not distinguishable by bottom-up proteomics. The presented workflow will facilitate proteoform-directed analysis on samples of limited availability.
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