Sperm lysates were extracted in 50 μL of lysis buffer (8 M Urea, 0.2 M Ammonium Bicarbonate) with sonication. Each sperm lysate was trypsin-digested and labeled with iTRAQ 4-plex reagent (AB SCIEX) according to the manufacture’s instruction. The labeled peptides were applied to QTRAP®4500 LC/MS/MS System (AB SCIEX). These experiments were performed in duplicate. Identification and quantification of proteins from sperm lysates were performed using the ProteinPilot software, Version 4.0 (AB SCIEX).
Proteinpilot software version 4
Manufactured by AB Sciex
ProteinPilot software version 4.5 is a core software tool for protein identification and quantification. It is designed to process and analyze data from mass spectrometry experiments.
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8 protocols using proteinpilot software version 4
1
Quantitative Proteomics of Sperm Lysates
2
Shotgun Proteomics Analysis of Mice
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The samples were resuspended in re-dissolving solution, loaded on a C18RP precolumn (100 μm x 3 cm, C18, 3 μm, 150 Å), and desalted for 10 min using the Eksigent nanoLC-Ultra™ 2D System (Eksigent Technologies, USA). Separation was performed using an elution gradient from 5 to 35% beginning with Eluant A (consisting of 94.9% deionized water, 5.0% methanol, and 0.1% formic acid, pH = 3) and transitioning to Eluant B (5.0% deionized water, 94.9% methanol, and 0.1% formic acid) over 70 min. The LC-MS/MS analysis was performed using a TripleTOF 5600 System (Applied Biosystems/ MDSSIEX), and the MS data were acquired in information-dependent acquisition mode. The data were processed using the mouse database of the Protein Pilot Software version 4.0 (AB SCIEX). The tolerances were specified as ±0.05 Da for peptides and ±0.05 Da for MS/ MS fragments. False discovery rate (FDR) analysis was also performed using the internal tools of ProteinPilot. Only the proteins identified with a local FDR ≤ 5% were considered for further analysis.
3
Quantitative Proteomic Analysis by iTRAQ
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Isobaric tags for relative and absolute quantification (iTRAQ), a chemical labeling mass spectrometry method, has been performed following the manufacturer’s protocol (AB SCIEX, Framingham, MA, USA) [19 (link), 20 (link)]. Protein identification and relative quantification were carried out using ProteinPilot Software Version 4.5 (AB SCIEX) [21 (link)]. Function definitions of the variable protein contents were searched against the Swissport database (Release, 10/16/2013). Protein ratios were normalized using the overall median ratio for all the peptides in the sample for each separate ratio in every individual experiment. A confidence cutoff for protein identification > 95% was applied. The specific pathway alteration was identified by Metacore (GeneGo, St. Joseph, MI) or KEGG ontology analysis (Kyoto University, Japan) [22 (link)].
4
Quantitative Phosphoproteomics of Hypoxia
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Total cell lysates were extracted from HeLa S3 cells that were cultured in normoxic or hypoxic conditions for 4–16 h. The protein extracts were digested with trypsin, and the peptides were labeled with iTRAQ reagent and enriched for phosphopeptides using iTRAQ Reagent-Multiple Assay Kit and Titansphere Phos-Tio Kit according to the manufacturer's instruction (Ab Sciex, Tokyo, Japan). Mass spectrometry analysis was performed on the iTRAQ-labeled extracts by using AB SCIE TripleTOF 5600 system and DiNa system (Filgen, Nagoya, Japan). The mass spectrometry spectra were extracted and searched for phosphorylation on Ser, Thy, and Tyr using Protein Pilot Software version 4.5 (Ab Sciex).
5
Proteomic Analysis of Exosome Labeling
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Proteomic analysis was performed as previously described [16 (link)]. In brief, exosomes were labeled with iTRAQ reagents using the iTRAQ multiplex kit (AB Sciex, Foster City, CA, USA). Labeled samples were separated and automatically spotted onto a MALDI plate using the direct nanoLC and MALDI fraction system DiNa-MaP (KYA Technologies, Tokyo Japan). Mass spectra were acquired using the AB Sciex TOF/TOF 5800 system operated on the TOF/TOF Series Explorer software version 4.1 (AB Sciex). All MS/MS data were submitted to the ProteinPilot software version 4.5 (AB Sciex). Protein identification was considered to be correct based on the following selection criteria: protein having at least 2 peptides with an ion score above 95% confidence; and protein with protein score (ProtScore) > 1.3 (unused, p < 0.05, 95% confidence).
6
Quantitative Proteomic Analysis of Exosomes
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For western blot analysis, total protein was extracted from exosomes, CRC cells, or mouse liver metastasis tissue using RIPA lysis buffer and analyzed as previously described (Fang et al., 2020 (link)). The exosomes were labeled with iTRAQ reagents using the iTRAQ multiplex kit (AB Sciex, USA) and followed analyzed according to previous publication (Kawakami et al., 2017 (link)). Labeled samples were separated and automatically spotted onto a MALDI plate, and mass spectra were acquired using the AB Sciex TOF/TOF 5800 system. All MS/MS data were analyzed via MASCOT and the Protein Pilot software (version 4.5; AB Sciex) to identify and quantify corresponding proteins in different groups (Supplementary Table S2 ). Protein identification was considered correct, based on the selection criteria (Kawakami et al., 2017 (link)).
7
Shotgun MS Data Analysis Protocol
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ProteinPilot® software version 4.5 (AB SCIEX) was used to analyze the shotgun MS data. The MS data were collectively queried against the predicted ORFs. A false discovery rate analysis in the ProteinPilot® software was performed and p-value threshold was set to 0.05. The sequences identified to be present in the sample tissue were annotated via a two-step homology search against Per40 DB and NR database using BLASTP as mentioned above (see Section 2.3.4 ).
8
Quantitative Proteomics Using iTRAQ
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Protein identification and quantification of iTRAQ data were performed using ProteinPilot software version 4.5 (AB SCIEX). The paragon algorithm in ProteinPilot software was used for peptide identification and isoform-specific quantification. The signal from the iTRAQ114 group served as the internal reference for signal intensity to which all signals were normalized. The weighted average of the ratios of the respective peptides was calculated based on the protein quantitation results. False discovery rate analysis was conducted, and the detected protein threshold was set to <0.01. The proteomic database used in this study was for Homo sapiens (UniProKB) (http://www.uniprot.org/uniprot ).
The differentially expressed proteins were set as follows: ≥1.2 indicating upregulated (P<0.05) and ≤0.8 indicating downregulated (P<0.05).
The differentially expressed proteins were set as follows: ≥1.2 indicating upregulated (P<0.05) and ≤0.8 indicating downregulated (P<0.05).
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