Development of an All-in-One Protein Digestion Platform Using Sorbent-Attached Membrane Funnel-Based Spray Ionization Mass Spectrometry
- Journal Article
In this work, the sorbent-attached microfunnels used in funnel-based spray ionization mass spectrometry were evaluated for the all-in-one digestion of proteins. Sorbent materials, including C18 and TiO2 powders, were used as substrates to support in-funnel digestion and subsequent solid-phase extraction and purification of the digested products. In-funnel digestion protocols with and without reductive alkylation were developed for the analysis of proteins with and without disulfide linkages. Compared with in-solution digestion of the same loadings, the sequence coverage of in-funnel digestion of ovalbumin (with one disulfide bond) and ovocystatin (with two disulfide bonds) increased from 36% to 65% and from 21% to 81%, respectively. Loading 100 fmol of ovalbumin was sufficient to generate detectable tryptic fragments on C18-attached funnels. Notably, some phosphorylated digestion fragments were solely detected on C18-attached funnels and some nonphosphorylated digestion fragments were detected only on TiO2-attached funnels. Complex biological protein mixtures (i.e., bovine milk) and mouse liver protein extract could also be digested on C18- and TiO2-attached funnels. Using this platform, 30 samples were digested at the same time with enhanced digestion efficiency and were analyzed by funnel-based spray ionization mass spectrometry. This approach is potentially useful for sensitive and high-throughput bottom-up proteomic studies of complex biological samples. © 2020 American Society for Mass Spectrometry. Show more
Journal / seriesJournal of the American Society for Mass Spectrometry
Pages / Article No.
PublisherAmerican Chemical Society
SubjectHigh-throughput; All-in-one protein digestion; Mass spectrometry; Mouse liver; Milk
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