Samples were prepared for mass spectrometry and analyzed as previously described (Stroud et al., 2016). Briefly, cells were lysed in 1% (w/v) sodium deoxycholate, 100 mM Tris-HCl (pH 8.1), Tris(2-carboxyethy)phosphine (TCEP), 20 mM chloroacetamide, and incubated at 99 °C for 10 min. Reduced and alkylated proteins were digested into peptides using trypsin by incubation at 37 °C overnight, according to the manufacturer’s instructions (Promega). Detergent was removed from the peptides using SDB-RPS stage tips as described (Stroud et al., 2016). Peptides were reconstituted in 0.1%% trifluoroacetic acid (TFA), 2% ACN, and analyzed by online nano-HPLC/electrospray ionization-MS/MS on a Q Exactive Plus connected to an Ultimate 3000 HPLC (Thermo Fisher Scientific). Peptides were loaded onto a trap column (Acclaim C18 PepMap nano Trap × 2 cm, 100 μm I.D, 5 μm particle size, and 300 Å pore size; Thermo Fisher Scientific) at 15 μl/min for 3 min before switching the pre-column in line with the analytical column (Acclaim RSLC C18 PepMap Acclaim RSLC nanocolumn 75 μm × 50 cm, PepMap100 C18, 3 μm particle size 100 Å pore size; Thermo Fisher Scientific). The separation of peptides was performed at 250 nl/min using a nonlinear ACN gradient of buffer A (0.1% FA, 2% ACN) and buffer B (0.1% FA, 80% ACN), starting at 2.5% buffer B to 35.4% followed by ramp to 99% over 278 min. Data were collected in positive mode using Data Dependent Acquisition using m/z 375–1575 as MS scan range, HCD for MS/MS of the 12 most intense ions with z ≥ 2. Other instrument parameters were MS1 scan at 70,000 resolution (at 200 m/z), MS maximum injection time 54 ms, AGC target 3E6, normalized collision energy was at 27% energy, isolation window of 1.8 Da, MS/MS resolution 17,500, MS/MS AGC target of 2E5, MS/MS maximum injection time 54 ms, minimum intensity was set at 2E3, and dynamic exclusion was set to 15 s. Thermo raw files were processed using the MaxQuant platform (Tyanova et al., 2016) version 1.6.5.0 using default settings for a label-free experiment with the following changes. The search database was the UniProt human database containing reviewed canonical sequences (June 2019) and a database containing common contaminants. ‘Match between runs’ was enabled with default settings. Maxquant output (proteinGroups.txt) was processed using Perseus (Tyanova et al., 2016) version 1.6.7.0. Briefly, identifications marked ‘Only identified by site,’ ‘Reverse,’ and ‘Potential Contaminant’ were removed along with identifications made using <2 unique peptides. Log2 transformed LFQ Intensity values were grouped into control and knockout groups, each consisting of three replicates. Proteins not quantified in at least two replicates from each group were removed from the analysis. Annotations (Gene Ontology Biological Process [GOBP]) were loaded through matching with the majority protein ID. A two-sample, two-sided t-test was performed on the values with significance determined using permutation-based FDR statistics (FDR 5%, S0 = 1). Enrichment analysis of GOBP terms was performed on significantly altered proteins using a significance threshold of 4% FDR.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.