期刊
JOURNAL OF PROTEOME RESEARCH
卷 18, 期 7, 页码 2915-2924出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.9b00217
关键词
bottom-up proteomics; top-down proteomics; intact mass spectrometry; universal solid-phase protein preparation (USP3)
Selecting a sample preparation strategy for mass spectrometrybased proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP3) method which is rapid, robust, and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis. This technique builds upon the single-pot solid-phase-enhanced sample preparation (SP3) where we now demonstrate its scalability (low to high micrograms of protein) and the influence of variables such as bead and enzyme amounts on the efficiency of protein digestion. We also incorporate acid hydrolysis of DNA and RNA during complete proteome extraction resulting in a more reliable method that is simple and easy to implement for routine and high-throughput analysis of proteins. We benchmarked the performance of this technique against filter-aided sample preparation (FASP) using 30 mu g of total HeLa protein lysate. We also show that the USP3 method is compatible with top-down MS where we reproducibly detect over 1800 proteoforms from 50 mu g of HeLa protein lysate. The USP3 protocol allows for efficient and reproducible data to be generated in a cost-effective and robust manner with minimal down time between sample collection and analysis by MS.
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