Global bottom-up proteomics workflow using Azo and PASEF on the timsTOF Pro. (A) Sample preparation, including (1) tissue cutting (scalable down to 1 mg of tissue), (2) tissue homogenization in Azo, a photocleavable surfactant, (3) rapid tryptic digest (as quick as 30 min), and (4) Azo degradation and C18 desalting prior to (5) RPLC-TIMS-MS/MS. (B) Schematic of PASEF on the timsTOF Pro, demonstrating parallel accumulation of ions, rapid quadrupole mass filter switching, and serial ion fragmentation.