Indicated bands from hr‐CN PAGE or BN‐PAGE were enzymatically digested, separated on a HPLC EkspertnanoLC 425 (Eksigent, Redwood City CA) and analyzed in a MALDI‐TOF/TOF 4800 Plus mass spectrometer (ABSciex, Framingham MA) (Shevchenko, Tomas, Havli, Olsen, & Mann, 2006 ) in the Unidad de Genómica, Proteómica y Metabolómica, CINVESTAV‐IPN. Generated MS/MS spectra were compared using Protein Pilot software v. 4.0 (ABSciex, Framingham MA) against the Saccharomyces cerevisiae ATCC 204508 database (downloaded of Uniprot, 6721 protein sequences) and Wolbachia genus database (downloaded of Uniprot, 47781 protein sequences) using Paragon algorithm.
Proteinpilot software v 4
Manufactured by AB Sciex
Sourced in United States, Canada
The ProteinPilot Software v.4.5 is a data analysis software designed for the identification and quantification of proteins from mass spectrometry data. The software provides a suite of tools for processing, analyzing, and interpreting complex proteomic data.
Automatically generated - may contain errors
Lab products found in correlation
Mascot v2, by Matrix Science (3 mentions)
Proteinpilot, by AB Sciex (2 mentions)
Mascot, by Matrix Science (2 mentions)
Tripletof 5600, by AB Sciex (2 mentions)
Ekspert nanolc425, by AB Sciex (1 mentions)
4800 plus maldi tof tof mass spectrometer, by AB Sciex (1 mentions)
Pro group algorithm, by AB Sciex (1 mentions)
Tripletof 5600 mass, by AB Sciex (1 mentions)
Analyst tf 1, by AB Sciex (1 mentions)
Trypsin, by Merck Group (1 mentions)
Eksigent nanolc ultra 2d system, by AB Sciex (1 mentions)
Eksigent nanolc system, by AB Sciex (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Mascot, by AB Sciex (1 mentions)
27 protocols using proteinpilot software v 4
1
Proteomic Analysis of Yeast and Wolbachia
2
Cucumber Proteome Quantification by MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mass spectrometric data was processed with Protein Pilot Software v. 4.0 (AB SCIEX, USA) against Cucumber database using the Paragon algorithm, and further processed by a Pro Group algorithm where isoform-specific quantification was adopted to trace the differences between expressions of various isoforms which was applied to the peptide identification. Protein identification was performed with emphasis on biological modifications option. Database search parameters were the followings: instrument was TripleTOF 5600, iTRAQ 8-plex quantification, cysteine modified with iodoacetamide, biological modifications were selected as the ID focus, trypsin digestion. An automatic decoy database search strategy was employed to estimate the false discovery rate (FDR) using the Proteomics System Performance Evaluation Pipeline Software (PSPEP) t was integrated in the Protein Pilot Software. In this study, only protein quantification data with the value of global FDR ≤0.05 were chosen for further analysis, and proteins with a |fold change ≥1.5| were considered to be significantly differentially expressed.
3
Quantitative Proteomics by iTRAQ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein identification and quantification were carried out using ProteinPilot™ software v.4.0 (ABSciex). Each MS/MS spectrum was searched in the Uniprot/Swissprot database (downloaded in May 2012) for Homo sapiens. Search parameters within ProteinPilot were set with trypsin cleavage specificity; methyl methanethiosulfate (MMTS) modified cysteine as fixed modifications; biological modification “ID focus” settings, and a protein minimum confidence score of 95%. Thus, the identity of the protein from the analyzed peptide was confirmed, and the ratios of the peak areas of iTRAQ reporter ions were used to compare the relative abundance of the protein identified in the sample. Only proteins identified with at least 95% confidence, or a Prot Score (protein confidence measure) of at least 1.3 were reported (Supplementary Table S1 ). Data were normalized for loading error by bias and the background correction was calculated using the Pro Group™ algorithm (ABSciex). To determine the cutoff value for significant fold changes, coefficient of variation and cumulative frequency were calculated for the common proteins employing the R statistical package58 (link). Using this tool, we considered statistically significant only those changes with a p value ≤ 0.05 and a ratio ≥ 1.4 (or ≤0.7). The results obtained from ProteinPilot were exported to Microsoft Excel for further analyses.
4
Shotgun Proteomics of Mouse Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DDA files were searched using Protein Pilot software v. 4.2 (Sciex, Concord, ON, Canada) and Mascot v. 2.4 (Matrix Science Inc., Boston, MA, United States). Trypsin as digestion enzyme was specified for both the software. For Mascot we used 2 missed cleavages, the instrument was set to ESI-QUAD-TOF and the following modifications were specified for the search: carbamidomethyl cysteine as fixed modification and oxidized methionine as variable modification. A search tolerance of 50 ppm was specified for the peptide mass tolerance, and 0.1 Da for the MS/MS tolerance. The charges of the peptides to search for were set to 2+, 3+, and 4+, and the search was set on monoisotopic mass.
The UniProt Swiss-Prot reviewed database containing mouse proteins (version 20july15, containing 23,304 sequence entries) was used and a target-decoy database search was performed. False Discovery Rate was fixed at 1%.
The UniProt Swiss-Prot reviewed database containing mouse proteins (version 20july15, containing 23,304 sequence entries) was used and a target-decoy database search was performed. False Discovery Rate was fixed at 1%.
5
Identification of Protein Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DDA files were searched using Protein Pilot software v. 4.2 (SCIEX, Concord, Canada) and Mascot v. 2.4 (Matrix Science Inc., Boston, USA). Trypsin as digestion enzyme was specified for both software. For Mascot we used 2 missed cleavages, set the instrument to ESI-QUAD-TOF and specified the following modifications for the assay: carbamidomethyl cysteine as fixed modification and oxidized methionine as variable modification. An assay tolerance of 50 ppm was specified for peptide mass tolerance, and 0.1 Da for MS/MS tolerance. The peptide charges to be detected were set to 2 +, 3 + and 4 +, and the assay was set on monoisotopic mass [21] . The UniProt Swiss-Prot reviewed database containing human proteins (version 2015.07.07, containing 42,131 sequence entries) was used and a target-decoy database search was performed. False Discovery Rate was fixed at 1%.
6
Mouse Protein Identification by Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mass spectrometry files were searched using Protein Pilot (AB SCIEX, Concord, Canada) and Mascot (Matrix Science Inc., Boston, USA). Samples were input in the Protein Pilot software v. 4.2 (AB SCIEX, Concord, Canada), which employs the Paragon algorithm, with the following parameters: cysteine alkylation, digestion by trypsin, no special factors and false discovery rate (FDR) at 1%. The UniProt Swiss-Prot reviewed database containing mouse proteins (version 20july15, containing 23,304 sequence entries). The Mascot search was performed on Mascot v. 2.4, the digestion enzyme selected was trypsin, with 2 missed cleavages and a search tolerance of 50 ppm was specified for the peptide mass tolerance, and 0.1 Da for the MS/MS tolerance. The charges of the peptides to search for were set to 2+, 3+ and 4+, and the search was set on monoisotopic mass. The instrument was set to ESI-QUAD-TOF (electrospray ionization quadrupole time-of-flight) and the following modifications were specified for the search: carbamidomethyl cysteines as fixed modification and oxidized methionine as variable modification.
7
Peptide Identification from DDA and DIA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DDA files were searched using Protein Pilot software v. 4.2 (AB SCIEX, Concord, ON, Canada) and Mascot v. 2.4 (Matrix Science Inc., Boston, MA, USA). The DIA files were converted to pseudo-MS/MS spectra with DIA-Umpire software and they were searched as DDA files [23 (link),24 (link)]. Trypsin as digestion enzyme was specified for both the software. For Mascot we used two missed cleavages, the instrument was set to ESI-QUAD-TOF, and the following modifications were specified for the search: carbamidomethyl cysteins as fixed modification and oxidized methionine as variable modification. A search tolerance of 0.08 Da was specified for the peptide mass tolerance, and 10 ppm for the MS/MS tolerance. The charges of the peptides to search for were set to 2 +, 3 +, and 4 +, and the search was set on monoisotopic mass. The UniProt Swiss-Prot reviewed database containing human proteins (version 2015.07.07, containing 42131 sequence entries) was used and a target-decoy database search was performed. False Discovery Rate was fixed at 1%.
8
Mass Spectrometry-Based Proteomics Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dried peptides were dissolved in 0.1% formic acid/2% acetonitrile and analyzed on AB Sciex Triple TOF 5600 mass spectrometer (MS) system. Acquired raw data files (.wiff) were searched using Protein Pilot software V.4.2 (AB Sciex, USA) against protein sequence database. Finally, obtained data were uploaded to IPA (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/ ) for bioinformatics analysis. As SILAC is a highly accurate quantitative method, cutoff for fold change was set to 1.50, and p-value was less than 0.05. This study employed global functional analyses, network analyses, and canonical pathway analyses.
For differentially expressed global protein samples, peptide sample of each gel piece was individually analyzed on MS. After searching against protein sequence database, nine sets of data were merged. Above experimental process was repeated thrice; data were merged and then uploaded to IPA for analysis.
For differentially expressed global protein samples, peptide sample of each gel piece was individually analyzed on MS. After searching against protein sequence database, nine sets of data were merged. Above experimental process was repeated thrice; data were merged and then uploaded to IPA for analysis.
9
In-Gel Tryptic Digestion and LC-MS/MS Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From the image analysis, spots of interest were cut and digested in-gel with 20 ug/mL trypsin (Sigma) at 37 °C O/N for liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The LC–MS/MS analyses were performed by a micro-LC Eksigent Technologies (Dublin, USA) system that included a micro LC200 Eksigent pump with flow module 5–50 μL, a programmable autosampler CTC PAL with a Peltier unit (1-45 °C). The LC system was interfaced with a 5600+ TripleTOFTM system (AB Sciex, Concord, Canada) equipped with DuoSprayTM Ion Source and CDS (Calibrant Delivery System). The data was acquired with Analyst TF 1.7 (AB SCIEX, Concord, Canada). Files were searched using ProteinPilot software v. 4.2 (ABSciex) with the Paragon algorithm. Protein spots were analyzed with the following parameters taken into consideration: cysteine alkylation, digestion by trypsin, no special factors and False Discovery Rate of 1%. Sequence identification was done using UniProt Swiss-Prot database containing human proteins (version 2015.06.09 reviewed, containing 20,207 sequence entries).
10
Quantitative Proteomic Analysis of HIV-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All DDA mass spectrometry
files were searched in unison using ProteinPilot software v. 4.2 (AB
Sciex) with the Paragon algorithm. Samples were input as unlabeled
samples with the following parameters: methyl methanethiosulfonate
(MMTS) cysteine alkylation, digestion by trypsin and no special factors.
The search was conducted using a through identification effort of
a UniProt Swiss-Prot database (November 2012 release) containing human
and HIV-1 proteins, as well as common laboratory contaminants. False
discovery rate analysis was also performed. The output of this search
is a .group file and was used as the MDM reference spectral library.
The .group file contains the following information that is required
for targeted data extraction: protein name and UniProt accession,
stripped peptide sequence, modified peptide sequence, Q1 and Q3 ion
detection, retention time, relative intensity, precursor charge, fragment
type, score, confidence, and decoy result.
files were searched in unison using ProteinPilot software v. 4.2 (AB
Sciex) with the Paragon algorithm. Samples were input as unlabeled
samples with the following parameters: methyl methanethiosulfonate
(MMTS) cysteine alkylation, digestion by trypsin and no special factors.
The search was conducted using a through identification effort of
a UniProt Swiss-Prot database (November 2012 release) containing human
and HIV-1 proteins, as well as common laboratory contaminants. False
discovery rate analysis was also performed. The output of this search
is a .group file and was used as the MDM reference spectral library.
The .group file contains the following information that is required
for targeted data extraction: protein name and UniProt accession,
stripped peptide sequence, modified peptide sequence, Q1 and Q3 ion
detection, retention time, relative intensity, precursor charge, fragment
type, score, confidence, and decoy result.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!