Computational protocol: Nucleolar expansion and elevated protein translation in premature aging

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Protocol publication

[…] Samples were denatured in 8 M urea/100 mM TEAB, pH 8.5; reduced with TCEP; alkylated with chloroacetamide; and digested overnight with trypsin. Digestion was quenched with 5% formic acid. Detergent was removed from pulse-labeled SILAC samples with SCX tips. Samples corresponding to SILAC analyses appearing in Fig. , Fig. , and Supplemental Fig.  were run on a Thermo Orbitrap Fusion Tribrid MS/MS with CID fragmentation. The digest was injected directly onto a 30 cm, 75 um ID column packed with BEH 1.7 um C18 resin. Samples were separated at a flow rate of 200 nl/min on a nLC 1000. Buffer A and B were 0.1% formic acid in water and acetonitrile, respectively. A gradient of 1–25%B over 160 min, an increase to 35%B over 60 min, an increase to 90%B over another 10 min and held at 90%B for a final 10 min of washing was used for 240 min total run time. Column was re-equilibrated with 20 ul of buffer A prior to the injection of sample. Peptides were eluted directly from the tip of the column and nanosprayed directly into the mass spectrometer by application of 2.5 kV voltage at the back of the column. The Orbitrap Fusion was operated in a data dependent mode. Full MS scans were collected in the Orbitrap at 120 K resolution with a mass range of 400 to 1500 m/z and an AGC target of 4e. The cycle time was set to 3 s, and within this 3 s the most abundant ions per scan were selected for CID MS/MS in the ion trap with an AGC target of 1e and minimum intensity of 5000. Maximum fill times were set to 50 ms and 100 ms for MS and MS/MS scans respectively. Quadrupole isolation at 1.6 m/z was used, monoisotopic precursor selection was enabled, charge states of 2–7 were selected and dynamic exclusion was used with exclusion duration of 5 s.Samples corresponding to the SILAC analysis appearing in Fig.  were run on a Thermo Q-Exactive Quadrupole-Orbitrap MS/MS using previously optimized settings. The digest was injected directly into a 30 cm, 75 um ID column packed with BEH 1.7 um C18 resin. Samples were separated at a flow rate of 200 nl/min on an LC 1000. Buffer A and B were 0.1% formic acid in water and acetonitrile, respectively. A gradient of 5–30% B over 280 min, an increase to 40% B over 60 min, and increase to 90% B for another 10 min, followed by 90% B for the final 10 min was used for the 360 min total run time. Peptides were eluted directly from the tip of the column and nanosprayed directly into the mass spectrometer by application of 2.5 kV voltage at the back of the column. The Q Exactive was operated in data-dependent mode. Full MS1 scans were collected in the Orbitrap at 70 k resolution with a mass range of 400 to 1800 m/z and an AGC target of 5e. The ten most abundant ions per scan were selected for MS/MS analysis with HCD fragmentation of 25NCE, an AGC target of 5e and minimum intensity of 1e. Maximum fill times were set to 120 ms and 500 ms for MS and MS/MS scans respectively. Quadrupole isolation of 2.0 m/z was used, dynamic exclusion was set to 15 s and charge states of 1 and unassigned were excluded.Peptide and protein identification, quantification, and analysis were performed with Integrated Proteomics Pipeline-IP2 (Integrated Proteomics Applications; www.integratedproteomics.com). Tandem mass spectra were extracted from raw files using RawConverter and searched with ProLUCID against the human UniPROT database. The search space included all fully tryptic and half-tryptic peptide candidates. Carbamidomethylation on cysteine was considered as a static modification. Data were searched with 50 ppm precursor ion tolerance and 600 ppm fragment ion tolerance. Data was filtered to 10 ppm precursor ion tolerance post-search. Identified proteins were filtered using DTASelect and utilizing a target-decoy database search strategy to control the false discovery rate to 1% at the protein level. Census was utilized for quantitative analysis of SILAC labeled peptides. Peptide ratios were calculated for each tryptic peptide with a profile score >0.8 as the peak area ratio of the heavy isotope-containing mass spectrum to light isotope-containing mass spectrum. Ratios were averaged for all peptides identified for each protein. […]

Pipeline specifications

Software tools RawConverter, ProLuCID, DTASelect
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Progeria, Genetic Diseases, Inborn