Spectronaut pulsar x

From each fraction, 6 μL was removed and added to 2 μL of 10 × iRT standard peptide, and 2 μL of each sample was separated with nano-LC and analyzed by online electrospray tandem MS. The complete liquid-mass tandem system consisted of a liquid system (Waters Acquity UPLC; Waters, MA, USA) and an MS system (Q-Exactive HF; Thermo Fisher Scientific, MA, USA). Buffer A consisted of 0.1% formic acid aqueous solution, and buffer B consisted of 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 80%). The sample was separated by an analytical column (Thermo Fisher Scientific, MA, USA; Acclaim PepMap C18, 75 μm × 25 cm) at a flow rate of 200 nL/min with the following gradient: 0–5 min, 1% B; 5–95 min, 1% B to 28% B; 95–110 min, 28% B to 38% B; 110–115 min, 38% B to 100% B; and 115–120 min, 100% B. The electrospray voltage was 2.0 kV. The MS parameters were set as follows: (1) MS: scan range (m/z) = 350–1600, resolution = 60,000, AGC target = 3e6, maximum injection time = 50 ms and filter dynamic exclusion: exclusion duration = 30 s; and (2) dd-MS2: isolation window = 4 m/z, resolution = 15,000, AGC target = 5e5, maximum injection time = 80 ms and NCE = 30%. The MS raw data were analyzed and searched by Spectronaut Pulsar X (version 12, Biognosys AG), and a spectral database was established. The standard for library construction was 1% precursor FDR and 1% peptide FDR.

The raw MS data from DDA analysis were searched using Proteome Discoverer software (version 2.1; Thermo Fisher, Waltham, MA, United States) with SEQUEST HT against the SwissProt human database appended with the iRT fusion protein sequence. The parent ion tolerance was set at 10 ppm, and the fragment ion mass tolerance was set to 0.05 Da. A maximum of two missed cleavage sites in the trypsin digestion was allowed. Carbamidomethylation of cysteines was set as a fixed modification, and the oxidation of methionine was considered a variable modification. The false discovery rate (FDR) of protein identifications was accepted less than 1.0%. Then, the results were imported to Spectronaut Pulsar X (Biognosys AG, Switzerland) software to generate the spectra library.
Then, all the raw DIA files were imported into Spectronaut Pulsar X as default settings. The optimal XIC extraction window was determined according to the iRT calibration strategy. The mass tolerance strategy was set to dynamic based on the extensive mass calibration. The cross-run normalization was set to the local normalization based on the local regression. The sum peak areas of the respective fragment ions in MS₂ were used for the quantification of peptide intensities. The missing values of protein abundance were filled with the KNN method.

The raw data from DIA proteomics analysis were searched using Spectronaut Pulsar X (Biognosys, AG, Schlieren, Switzerland) software with default settings. The mass spectrometry proteomics data have been deposited at ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD031838. An optimal XIC extraction window was identified based on the iRT calibration strategy. The mass tolerance strategy was set to dynamic based on extensive mass calibration. The cross-run normalization was set to local normalization based on local regression. The sum peak areas of the respective fragment ions in MS₂ were used to quantify peptide intensities. The k-nearest neighbors (KNN) method was used to fill in missing values of protein abundance.

The raw data of DIA-MS was processed with Spectronaut Pulsar X (v12, Biognosys AG). After normalization, differentially expressed proteins between EBV-positive and negative GC were identified. The threshold were set as absolute fold change (FC) > 1.5 and P < 0.05 corrected with 1% false discovery rate (FDR). Protein–protein interaction (PPI) information was downloaded from the STRING online tool (v11.0, https://string-db.org) and PPI network was constructed with Cytoscape software (v3.6.1). Funrich database (v3.1.3) was applied to gene enrichment analysis including expression site, Gene Ontology (GO) and biological pathways. The online datasets of gene expression profiling by microarray about EBVaGC were searched in Gene Expression Omnibus (GEO) database and analyzed with GEO2R package. Data processing and mapping was performed using R-project (v4.0.3) and Rstudio software (v1.3.1093). SPSS (v22.0) software was employed to analyze the data of validation experiments, including χ² test, independent t test or Mann–Whitney U test, Kaplan–Meier test, log rank test and Cox regression, etc.. All the tests were judged as statistically significant when |FC| > 2.0 and P < 0.05 after correction with Benjamini-Hochberg (BH) method (FDR).

Mass spectrometry DIA and DDA raw data files acquired via the Orbitrap Fusion Lumos have been uploaded to the ProteomeXchange Consortium via the PRIDE partner repository (Perez‐Riverol et al., 2019) with the dataset identifier PXD017605 (Monroe et al., 2020). Spectral library files, the proteome FASTA file for A. polyacanthus, used to create these libraries, and raw data output from Spectronaut Pulsar X (Version 12, Biognosys) after quantitative analysis can also be found in this repository.

The DDA library creation required the injection of all fractions into the timsTOF mass spectrometer (Bruker) via an Evosep One system liquid chromatography. Protein samples were scanned with 8 cycles of parallel accumulation serial fragmentation (PASEF) MS/MS.
For DDA library data, the FASTA sequence database was searched with Spectronaut™ (Biognosys, version 14.4.200727.47784) software. The database was Uniprot_human database, iRT peptides sequence was added (Biognosys|iRTKit|). The parameters were set as follows: the enzyme was trypsin, max missed cleavages was 2, fixed modification is carbamidomethyl(C), dynamic modification is oxidation(M), and acetyl (Protein N-term). All reported data were based on 99% confidence for protein identification as determined by false discovery rate (FDR =N(decoy)*2/(N(decoy)+ N(target))) ≤ 1%. The spectral library was constructed by importing the original raw files and DDA searching results into Spectronaut Pulsar X™ (Biognosys, version 12.0.20491.4).
DIA data was analyzed with Spectronaut™ searching the above constructed spectral library. Main software parameters were set as follows: retention time prediction type is dynamic iRT, interference on MS2 level correction is enabled, and cross-run normalization is enabled. All results were filtered based on Q value cutoff 0.01 (equivalent to FDR<1%).

The DDA collection results of the above 10 components were imported into the Proteome Discoverer software (version 2.1, Thermo Scientific, Waltham, MA, USA) search database using the following parameters: mouse database (released in 2019, containing 17,038 sequences) with the iRT peptide sequence attached, trypsin digestion, a maximum of two missing cleavage sites, parent ion mass tolerance of 10 ppm, fragment ion mass tolerance of 0.02 Da, methionine oxidation set as variable modification, cysteine carbamidomethylation set as fixed modification, and protein false discovery rate (FDR) set to 1%. The PD search result was used to establish the DIA acquisition method, and the window width and number were calculated according to the m/z distribution density.
Sixty-nine peptide samples were put into DIA mode to collect mass spectrometry data. Spectronaut™ Pulsar X (Biognosys, Biognosis, Switzerland) software was used to process and analyse mass spectrometry data [14 (link)]. Based on the DDA search result pdResult file and the 10 DDA raw files, we created a spectrum library; the raw files collected by DIA were imported for each sample to search the library. The high-confidence protein standard was a peptide q value < 0.01, and the peak area of all fragment ions of the secondary peptide was used for protein quantification.