DOI: 10.25345/c5cf9jk5g
title: MassIVE MSV000098258 - AAV2 induced RPA exhaustion generates cellular DNA damage and restricts viral gene expression
title: In Gel Digestion Each lane sample was then cut into small rectangular pieces and divided into two fractions: fraction 2 containing the streptavidin band and fraction 1 containing the remaining proteins. For in gel digestion, both fractions were subjected to several rounds of washing to remove salts and Coomassie. Specifically, gel pieces were incubated for 15 min in 25 mM ammonium bicarbonate, 50% ethanol 60 C for strongly stained pieces, and the supernatant was discarded. This wash step was repeated 2 3 times or until gel pieces were destained completely. Next, in preparation for cysteine reduction & alkylation, fractions were incubated in 100% ethanol for 10 min to dehydrate the gel pieces. Cysteine residues were reduced by incubating fractions for 20 min at room temperature in 20 mM DTT, 50 mM ammonium bicarbonate. After discarding the supernatant, cysteine residues were alkylated by a 20 min incubation in 80 mM chloroacetamide, 50 mM ammonium bicarbonate. Alkylation compounds were removed with successive 10 min washes in i 50 mM ammonium bicarbonate, ii 25 mM ammonium bicarbonate, 50% ethanol, and iii 100% ethanol. Gel pieces were subsequently dried in a vacuum concentrator. For overnight digestion at 37 C, trypsin solution 0.25 ug trypsin Promega in 50 ul of 50 mM ammonium bicarbonate was added to each fraction. On the following day, peptides were extracted by two sequential 15 min elutions in 150 ul of elution solution 80% acetonitrile, 0.2% formic acid under sonication. The resulting eluates were pooled and evaporated to dryness in a vacuum concentrator. LC MS analysis For LC MS analysis, a Vanquish Neo UHPLC system Thermo Fisher Scientific was coupled to an Orbitrap Ascend mass spectrometer Thermo Fisher Scientific via a Nanospray Flex ionization source. A 40 cm in house prepared fused silica C18 column was maintained at 50 C using a custom built column heater. The source voltage was set to 2 kV, and the ion transfer tube was held at 275 C. Peptide samples from streptavidin containing fractions were injected at a total amount of 500 ng, whereas 1 ug of peptides was loaded for all other samples. Peptides were separated at a flow rate of 300 nL per min with a 73 min active gradient from 5% to 46% solvent B solvent A: 0.2% FA; solvent B: 80% acetonitrile with 0.2% FA. Orbitrap Ascend parameters were as follows: MS1 scans were acquired at a resolution of 240k scan range 300 1350 m per z, with a maximum injection time of 50 ms, an automatic gain control AGC target of 1 x 106, a normalized AGC target of 250%, and an RF lens percentage of 30. Data dependent acquisition employed a 1 s cycle time. MIPS monoisotopic peak determination was set to peptide, and the isolation window center was set to most abundant peak. Charge states 2 5 were included, and dynamic exclusion was set to 20 s with a 5 ppm mass tolerance. MS2 spectra were recorded in the ion trap using an isolation window of 0.8 m per z, a normalized HCD collision energy of 24%, an ion trap scan rate set to Turbo, a scan range of 150 1350 m per z, a normalized AGC target of 250%, and a maximum injection time of 12 ms. Proteomics data analysis RAW files generated from the LC MS runs were analyzed with MaxQuant version 2.4.2.0, searching against a UniProt human FASTA file containing both SwissProt and TrEMBL entries. Adeno associated virus 2 proteins Rep68 P03132 and Rep78 Q89268 were added to this database. Unless otherwise specified, all MaxQuant parameters were set to their default values. Gel fractions originating from the same lane were assigned identical experiment names but treated as separate fractions in MaxQuant. In the Group specific parameters tab, Type was set to multiplicity = 2, and Arg10 and Lys8 were checked. Under Misc., Re quantify was enabled. In the Global parameters tab, the FASTA files described above were specified under Sequences. Under Protein quantification, Label min. ratio count was set to 1, and under Identification, Min. unique peptides was set to 1 with Match between runs enabled. Finally, under Label free quantification, iBAQ was enabled.