Rapid, Specific N-terminal R/K for Enhanced Sensitivity, de novo, and Confidence. Tryp-N™, or “N-terminal trypsin”, is a specialized protease from ProtiFi™ that cleaves proteins at the N-terminal…

Reagents The following chemicals and reagents were used: Dounce homogenizer (DWK Life Sciences, 885302-0002); Pierce anti-HA magnetic beads (Thermo Scientific, 88837); Pierce anti-Flag magnetic agarose (Thermo Scientific, A36797); anti-Flag M2 magnetic beads (Sigma Millipore, M8823); Pierce protein A/G magnetic beads (Thermo Scientific, 88802); IGEPAL CA-630 (Sigma-Aldrich, I8896); S-Trap micro columns (Protifi, C02-micro-80); triethylammonium bicarbonate (TEAB) buffer (Sigma-Aldrich, T7408); sodium dodecyl sulfate (SDS; Bio-Rad, 1610302); DSSO (Thermo Scientific, A33545); DHSO (CF Plus Chemicals, PCL042); DMTMM (Sigma-Aldrich, 74104); n-dodecyl β-d-maltoside (DDM, Gold Biotechnologies, DDM5); NativeMark protein standard (Invitrogen, LC0725); NativePAGE 4–6% gels (Invitrogen, BN1002BOX); MultiScreen filter plates (Sigma Millipore, MSHVN4510); TMTpro 16plex set (Thermo Fisher Scientific, A44520); protease inhibitor cocktail (Roche, 4906845001); tris(2-carboxyethyl)phosphine (TCEP; Gold Biotechnology, 51805-45-9); 2-chloroacetamide (Sigma-Aldrich, C0267); S-methyl thiomethanesulfonate (MMTS; Sigma-Aldrich, 208795); trypsin (Promega, V511C); Lys-C (Wako Chemicals, 129-02541); hydroxylamine solution (Sigma-Aldrich, 438227); Sep-Pak C18 and C8 50 mg cartridge (Waters, WAT054955 and WAT054965); high-pH reversed-phase peptide fractionation kit (Thermo Scientific, 84868); Bio-Rad protein assay dye (Bio-Rad, 5000006); 3-propanesulfonic acid (Thermo Scientific, J61296AE); Empore SPE discs C18 (Sigma Millipore, 66883-U); Gateway LR Clonase II enzyme mix (Thermo Scientific, 11791020); NEBNext Ultra II Q5 Master Mix (New England BioLabs, M0544L); Cas9-NLS (QB3 MacroLab at University of California, Berkeley); CloneR (StemCell Technologies, 05889); MiSeq reagent nano kit v2 (300 cycles; Illumina, MS-103-1001); GeneArt Precision gRNA synthesis kit (Thermo Fisher Scientific, A29377); RNAeasy Qiagen kit (Qiagen, 74104); 24-well glass-bottom plates (Cellvis, P24-1.5H-N); Corning square culture dish (Corning, 431110); Nunc Nunclon Delta cell culture dishes (Thermo Scientific, 140675, 150318 and 168381); Corning Matrigel matrix (Corning, 354230); DMEM with F-12 (Gibco, 11330057); neurobasal medium (Thermo Scientific, 21103049); non-essential amino acids (Gibco, 11140050); GlutaMAX (Gibco, 35050061); N-2 supplement (Gibco, 17502048); neurotrophin-3 (NT3; Peprotech, 450-03); brain-derived neurotrophic factor (BDNF; Peprotech, 450-02); B27 (Gibco, 17504001); Y27632 dihydrochloride (ROCK inhibitor; PeproTech, 1293823); Cultrex 3D culture matrix laminin I (R&D Systems, 3446-005-01); accutase (StemCell Technologies, 07922); FGF2-G3 (in-house); human insulin (Santa Cruz Biotechnologies, sc-360248); transforming growth factor-β (PeproTech, 100-21); holo-transferrin human (Sigma-Aldrich, T0665); sodium bicarbonate (Sigma-Aldrich, S5761-500G); sodium selenite (Sigma-Aldrich, S5261-10G); doxycycline (Clontech Labs, 631311); UltraPure 0.5 M EDTA (Invitrogen, 15575020); 16% paraformaldehyde (Electron Microscopy Science, 15710); DMEM (Gibco, 11995073); fetal bovine serum (Cytiva, SH30910.03); hydrocortisone (Sigma-Aldrich, H0135); polyethylenimine (Polysciences, 23966); FuGENE (Promega, E2311).