2.3. Mass Spectrometry and Data Analysis

The mass spectra of the peptides were obtained with a data-dependent 4-event scan method (a survey Fourie Transfrom (FT)-Mass Spectrometry (MS) parent scans followed by FT-MS/MS tandem mass spectrometry). Protein identifications was carried out using Proteome Discoverer 1.2 software in combination with the SEQUEST protein database search engine and International Protein Index (IPI) Human Protein Database (version 1.79). A sequential database search was performed using the human FASTA database. Only peptides with a cross-correlation (XCorr) cutoff of 2.6 for [M + 2H]²⁺, 3.0 for [M + 3H]³⁺ and a higher charge state were considered. These SEQUEST criteria typically result in a 1%–2% false discovery rate (FDR). The FDR was determined by searching on a decoy database. We used SIEVE 2.1 software (Thermo Fisher, Waltham, MA, USA) for label-free quantitative analysis of the high resolution MS spectra produced by Orbitrap MS scans. We then explored protein networks in SMAPP1-treated cells using results from SIEVE 2.1 in Ingenuity Pathway Analysis (Qiagen, Valencia, CA, USA). Venn diagrams were constructed using Genevenn freeware (http://genevenn.sourceforge.net/).