NanoLC MS MS analysis Samples g of yeast

NanoLC-MS/MS analysis. Samples (2µg of yeast cell lysate+different spiked level of UPS1) were analyzed in triplicate by nanoLC-MS/MS using a nanoRS UHPLC system (Dionex, Amsterdam, The Netherlands) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). 2µL of each sample were loaded on a C-18 precolumn (300µm IDx5mm, Dionex) at 20µL/min in 5% acetonitrile, 0.05% TFA. After 5min desalting, the precolumn was switched online with the analytical C-18 column (75µm IDx15cm, in-house packed with C-18 Reprosil) equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using the following gradient of solvent B at 300nL/min flow rate: 5–25% gradient during 75min; 25–50% during 30min; 50–100% during 10min. The LTQ-Orbitrap Velos was operated in data-dependent acquisition mode with the XCalibur software. Survey scan MS were acquired in the Orbitrap on the 300–2000m/z range with the resolution set to a value of 60,000. The 20 most intense ions per survey scan were selected for CID fragmentation and the resulting fragments were analyzed in the linear trap (LTQ). Dynamic exclusion was employed within 60s to prevent repetitive selection of the same peptide. MS data processing. The dataset was processed according to different workflows listed in Table 1 from Ref. [1], consisting in the following steps: peaklist generation, database search, validation of the identified 5 alpha reductase and extraction of quantitative metric (spectral count or MS signal). According to the different tools used for each step, 8 distinct workflows were evaluated. The same databases were used for peptide identifications: yeast database from UniprotKB (S_cerevisiae_ 20121108.fasta, 7798 sequences) and a compiled database containing the UPS1 human sequences (48 sequences). Bioinformatic workflow 1: ExtractMSn/Mascot/MFPaQ/Spectral Counting. The Mascot Daemon software (version 2.4; Matrix Science, London, UK) was used to perform database searches, using the Extract_msn.exe macro provided with Xcalibur (version 2.0 SR2; Thermo Fisher Scientific) to generate peaklists. Parameters used for creation of the peaklists were: parent ions in the mass range 400–4500, no grouping of MS/MS scans, and threshold at 1000. Peaklists were submitted to Mascot database searches (version 2.4.2). ESI-TRAP was chosen as the instrument, trypsin/P as the enzyme and 2 missed cleavages were allowed. Precursor and fragment mass error tolerances were set at 5ppm and 0.8Da, respectively. Peptide variable modifications allowed during the search were: acetyl (Protein N-ter), oxidation (M), whereas carbamidomethyl (C) was set as fixed modification. To calculate the false discovery rate (FDR), the search was performed using the “decoy” option in Mascot. Validation was performed with an in-house developed module associated to MFPaQ [2] (http://mfpaq.sourceforge.net/), based on the target-decoy strategy, as described before [3]. Briefly, FDR at peptide level was calculated as described in [9] and set at 5% by adjusting peptide p-value threshold. Validated peptides were assembled into protein groups following the principle of parsimony (Occam׳s razor) [10]. Protein groups were then validated to obtain a FDR of 1% at the protein level, by adjusting the threshold on a protein group score defined as the sum of peptide score offsets (difference between each peptide Mascot score and its homology or identity threshold). The total spectral count metric was extracted for each protein group by MFPaQ in each analytical run. Workflow 2: Andromeda/MaxQuant/Spectral Counting. Acquired MS data were processed using MaxQuant version 1.3.0.5 [4]. Derived peak lists were submitted to the Andromeda search engine [6]) (www.maxquant.org). For database searches, the precursor mass tolerance was set to 20ppm for first searches and 6ppm for main Andromeda database searches. The fragment ion mass tolerance was set to 0.5Da. Trypsin/P was chosen as the enzyme and 2 missed cleavages were allowed. Oxidation of methionine and protein N-terminal acetylation were defined as variable modifications, and carbamidomethylation of cysteine was defined as a fixed modification. Minimum peptide length was set to six amino acids. Minimum number of unique peptides was set to one. Maximum FDR – calculated by employing a reverse database strategy – were set to 1% for peptides and proteins. Proteins identified as “reverse” and “only identified by site” were discarded from the list of identified proteins. In this particular workflow, total spectral count for each validated protein group was computed from msms.txt table.