High-pH Reversed-Phase Fractionated Neural Retina Proteome of Normal Growing C57BL/6 Mouse
Mouse was sacrificed at postnatal day 46 by cervical dislocation. Retina were prepared seamlessly with suspension trap (S-TRAP) followed by high-pH reversed-phase peptide fractionation. Peptides fractions were identified with data-dependent acquisition by quadrupole time-of-flight mass spectrometry.
Sample Processing Protocol
Mouse were sacrificed at postnatal day 46 by cervical dislocation. Neural retinas were dissected in phosphate-buffered saline (PBS), isolated and immersed to 150uL of extraction buffer: 5% SDS, 50mM TEAB, homogenized with 2mL homogenizing mixed beads kit (KT003961-1-009.2, Precellys) at 4oC, 5,500rpm for 30s, 4 cycles with 20s intervals (Precellys Evolution, Bertin Instruments). The concentration of each extract was determined by protein assay and normalized to 2ug/μL (Pierce Rapid Gold BCA Protein Assay Kit, A53225, Thermo Fisher). Protein extracts (100ug) were treated with 20mM of DTT at 95oC for 10min and followed by 40mM of IAA for 30min in room temperature in the dark. The mixture was acidified with 1.2% phosphoric acid, add 200uL of 90% MeOH, 0.1M TEAB. The mixture was placed into the S-Trap Micro Spin column in a 1.7mL Eppendorf tube and centrifuge the column at 4000g for 20s. Wash in triplicates with 150uL of 90% MeOH, 0.1M TEAB and centrifuge at 4000g for 20s. Pipette 20uL of digestion buffer containing trypsin at 1:25 (w/w, trypsin: protein) and store at 47oC for 1h. Peptides were eluted stepwise with 40uL of 50mM TEAB, 40uL of 0.2% formic acid and 35uL of 50% ACN, 0.2% FA by centrifuge at 4000g for 20s. Eluents were pool and dried with vacuum centrifuge at 4oC. Peptides were reconstituted in 0.1% FA. Peptides (100ug) were fractionated according to Pierce High-pH reversed-phase peptide fractionation protocol. In brief, peptide was diluted with 300uL of 0.1% TFA solution and eluted with 5%, 7.5%, 10%, 12.5%, 15% and 17.5% of ACN, 0.1% TEA solution. Fractions were dried and normalized to 0.5ug/μL with 0.1% FA solution.
Data Processing Protocol
Peptides (2ug) were loaded on a C18 trap column (5um, 100um x 20mm, Acclaim PepMap 100) at 2ul/min isocratic flow for 15min and separated on a C18 analytical column (5um, 75um x 150mm) with 120min gradient conditions from 3-40% ACN, 0.1% FA at a flow rate of 300nl/min with Eksigent nanoLC 415. Precursor selection was set as 350-1800 m/z. The top four abundant ions were isolated for fragmentation by collision induced dissociation (CID), with charge state 2-5, signal exceeds 125 cps, and rolling collision energy, operated in positive ion mode connected with a nanospray source in Sciex TripleTOF 6600 mass spectrometer. Generated MS spectra were searched against the UniProt Mus musculus proteome database (modified on 21 Apr 2019) for protein identification via the ProteinPilot (ver. 5.2).
Chuen Lam, The Hong Kong Polytechnic University/School of optometry
Chuen Lam, Associate Professor, Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong ( lab head )