3D-SISPROT: A simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology for deep proteome profiling

- Achieve protein digestion and three-dimension separation for deep proteome profiling.
- Integrate SAX-based and high-pH RP fractionation in a pipette tip for the first time.
- Protein digestion does not influence the SAX-based peptide fractionation efficiency.
- The SAX plus C18 membranes design minimizes the sample loss.
- 8222 Proteins are identified by 3D-SISPROT from 30 μg cell lysates with 20.4 h.

Multidimensional peptide fractionation strategies have been approved as the efficient approaches to significantly improve the depth of proteome coverage. In this study, a simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology (3D-SISPROT) was developed for the deep proteome profiling from low microgram of proteins as starting materials. All the sample preparation steps, including protein digestion, strong anion exchange (SAX)-based fractionation, and high-pH reversed phase (RP) fractionation were integrated into one pipette tip packed with SAX and C18 membranes for the first time. The SAX plus C18 membranes design minimizes the sample loss and ensures high efficient SAX-based digestion. 4275 proteins were identified with 1.4 h of MS time when 6 μg cell lysates was processed. More importantly, the SAX-based digestion procedure did not influence the SAX-based peptide fractionation efficiency which was done in the same SAX membrane. The 3D-SISPROT was exemplified by the analysis of 30 μg of HEK 293T cell lysates with 20.4 h of MS time, which resulted in the identification of 8222 proteins including 3215 annotated membrane proteins. Gene Ontology annotations indicated that the 3D-SISPROT was unbiased for the proteins from major cellular components. Taking advantages of the efficient SAX-based and high-pH RP-based fractionation strategies, we expect that the 3D-SISPROT can be applied for the deep proteome profiling with limited starting material.