Development of an All-in-One Protein Digestion Platform Using Sorbent-Attached Membrane Funnel-Based Spray Ionization...

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.