Analysis of Mass Spectrometric Data of Proteins from Serum in Patients with Bipolar Disorder and and ealthy Individuals Using the PeptideShaker Software
Creators: name: Seregin, Alexander
name: Smirnova, Liudmila
name: Dmitrieva, Elena
name: Zavialova, Maria
name: Simutkin, German
name: Ivanova, Svetlana
Publisher: name: figshare
Year: 2023
Subjects: classificationCode: 320899
subject: Medical physiology not elsewhere classified
classificationCode: 320506
subject: Medical biochemistry - proteins and peptides (incl. medical proteomics)

description: In this work, the sera of patients with bipolar disorder and healthy people were purified by affinity chromatography from 14 major proteins and separated using 1D SDS-PAGE. After trypsinolysis, the resulting samples were identified by HPLC/mass spectrometry using an Ultimate 3000 Nano LC HPLC system (Thermo Scientific, Rockwell, IL, USA) coupled to a Q Exactive HF-X hybrid quadrupole - Orbitrap (Thermo Fisher Scientific).Peptide separation was performed on a C18 column with an inner diameter of 75 μm and a length of 150 mm (Acclaim® PepMap™ RSLC, Thermo Fisher Scientific, Rockwell, IL, USA). One microliter of sample, equivalent to one microgram of peptides, was loaded directly onto the column and equilibrated isocratically with mobile phase C (2% acetonitrile, 0.1% formic acid). The peptides were then eluted using a linear gradient of 5 to 55% buffer B (0.1% formic acid and 80% acetonitrile) at a flow rate of 0.3 μl/min. for 75 minutes, then for another 6 minutes to reach 99% buffer B. Before the next sample was loaded, a 10-minute wash with 99% buffer B and a 7-minute re-equilibration of the column with buffer A (0.1% formic acid) was performed.Then, the eluting peptides were loaded into the mass spectrometer through a capillary at a temperature of 240°C and an emitter voltage of 2.1 kV.Samples were analyzed in triplicate in Full MS mode followed by a single DDA MS2. Mass spectra were obtained in the mass range 320–1500 m/z with a resolution of 120,000 (MS). Precursor ions were fragmented in the HCD mode (Higher-Energy Collision Dissociation). Tandem mass spectra of the fragments were obtained at a resolution of 15,000 (MS/MS) in the range from 140 m/z to the m/z value determined by the charge state of the precursor, but not more than 2000 m/z. The maximum accumulation time of precursor ions was 50 ms, and that of fragment ions was 100 ms. The target fill value of the automatic gain control (AGC) for precursor and fragment ions was set to 1 x 10<sup>6</sup> and 2 x 10<sup>5</sup>, respectively.An isolation intensity threshold of 50,000 arbitrary units was defined for precursor selection, and up to 20 of the best precursors were selected for fragmentation at a normalized collision energy (NCE) of 30. The precursor ion isolation width was 2 m/z. Precursors with a charge state of 1+ and more than 5+ were rejected, and all measured precursors were dynamically excluded from the launch of the subsequent MS/MS for 20 s.Data processing was performed using the search algorithms Peptide Shaker version 2.1.9, OMSSA, X!Tandem, Andromeda, and the UniProtKB database. The search was carried out using SearchGUI version 3.3.12.Protein identification was conducted against a concatenated target/decoy version of the UniProtKB (version of v04/2019; 20303 (target) sequences) database considering the species Homo sapiens (40606 sequences). The decoy sequences were created by reversing the target sequences in SearchGUI. The identification settings were as follows: Trypsin, Specific, with a maximum of 1 missed cleavages 10.0 ppm as MS1 and 0.5 Da as MS2 tolerances; variable modifications: deamidation of N (+0.984016 Da), deamidation of Q (+0.984016 Da), oxidation of M (+15.994915 Da), oropionamide of C (+71.037114 Da); variable modifications during the refinement procedure: acetylation of protein N-term (+42.010565 Da), pyrolidone from E (-18.010565 Da), pyrolidone from Q (-17.026549 Da), pyrolidone from carbamidomethylated C (-17.026549 Da). The rest of the parameters were set by default.Peptides and proteins were inferred from the spectrum identification results using PeptideShaker version 2.2.9. Peptide Spectrum Matches (PSMs), peptides, and proteins were validated at a 1.0% False Discovery Rate (FDR) estimated using the decoy hit distribution. Proteins were considered as reliably identified if at least two of their peptides could be identified. Post-translational modification localizations were scored using the D-score and the phosphoRS score with a threshold of 95.0 as implemented in the compomics-utilities package. A phosphoRS score above this threshold was considered a confident localization.An unlabeled analysis based on an exponentially modified protein abundance index was used to detect differences in the relative abundance of detected proteins between study groups, e.g., emPAI (emPAI = 10 PAI <sup>-1</sup>). This index showed good specificity and sensitivity for samples with low protein content, which may be useful for determining minor proteins in blood serum The emPAI intensities for proteins were taken log2 and normalized to ensure equal mean protein content in all samples. Statistically significant differences in mean emPAI for each protein in the study group were assessed using a two-tailed unpaired Student's t-test (p=0.05).