MassIVE Reanalysis - RMSV000000269.1

RPXD017548.1

DIA-BGS-DIA using project-specific library : paired t-test

Description

Controlled quantitative mixtures : UPS2 protein standard spike-in into mouse cerebellum samples. The UPS2 was spiked into 10 biologically different cerebellum samples to simulate a real biological experiment with varying biological background. The concentration of the cerebellum samples was adjusted to 1 μg/μL and spiked with 5 different concentrations of the UPS2 digest in duplicates. The UPS2 consisted of 48 proteins organized in 6 abundance tiers each with 8 proteins, covering 5 orders of magnitude. On the basis of the lowest abundant tier the protein concentrations were S1: 0.05 amol/μL, S2: 0.055 amol/μL (+10%), S3: 0.072 amol/μL (+30%), S4: 0.136 amol/μL (+90%), and S5: 0.503 amol/μL(+270%) (assuming no losses during sample preparation). The sample set was measured in Biognosys facility (BGS). For library generation, 20 μg aliquots of the 10 samples were pooled. After fractionation, the pooled fractions were analyzed by a DDA method. DDA data were searched with MaxQuant 1.5.6.537 against the UniProt mouse database (downloaded on Jan sixth, 2016 as FASTA file) and the UPS2 database (www.sigma.com/ups) using the following settings, fixed modifications: carbamidomethyl (C); variable modifications: oxidation (M), acetyl (protein Nterm); enzyme: Trypsin/P; max. missed cleavages: 2; first search peptide tolerance: 20 ppm; main search peptide tolerance: 4.5 ppm; second peptide search was enabled. All other settings were set to default. Results were filtered by 1% FDR on PSM and protein level. The MaxQuant search results were imported into Spectronaut Pulsar 11 (11.0.15038.5)11 to generate a library using the default settings. To avoid interferences from peptides that are shared between the UPS2 proteins and the mouse proteome, these peptides were removed from the library using in house R scripts. DIA data were analyzed in Spectronaut Pulsar X (12.0.20228.0, Biognosys, Schlieren, Switzerland) using the previously generated library and default settings. The default normalization strategy was applied (local regression normal- ization38). The results were filtered by an FDR of 1% on precursor and protein group level (Q value <0.01). For statistical analysis, precursors with more than 20% missing values across the 10 MS runs ofDIA data were filtered out. The remaining precursors were mapped to protein IDs. All the intensities of the precursors uniquely mapped to a protein were summarized into a single protein-level intensity in each sample, by calculating the median intensity of precursors of the protein in a sample. The two-sample t-test was used separately for each protein to detect changes in protein abundance between two groups with the different spiked-in concentrations that are more systematic than as expected by random chance. description.pdf including details for data processing and statistical analysis and R script are available in the 'Methods' folder. [doi:10.25345/C5TJ1V]

[See results attachment job for details]

Keywords: MassIVE.quant reviewed - Platinum

Reanalyzed Datasets

  • MSV000084859 : Comparison of DIA and TMT based protein quantification in complex background
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Experimental Design
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    Biological Replicates:
    Technical Replicates:
 
Identification Results
    Proteins (Human, Remapped):
    Proteins (Reported):
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    Variant Peptides:
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Quantification Results
    Differential Proteins:
    Quantified Proteins:
 
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Species

Instrument

Modifications
Number of distinct conditions analyzed in this reanalysis.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this reanalysis.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates in this reanalysis.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this reanalysis.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates in this reanalysis.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed in files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported in this reanalysis.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported in this reanalysis.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported in this reanalysis.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported in this reanalysis.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified in this reanalysis.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this reanalysis.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison in this reanalysis.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this reanalysis.

"N/A" means no results of this type were submitted.