MassIVE Reanalysis - RMSV000000270.1

TMT with 10 fractions - BGS : 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. 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 ug/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) by TMT method. TMT : The spiked samples were labeled with the 10plex TMT kit. The combined TMT sample (200 μg cerebellum sample and 145 pmol of UPS2) was fractionated into 10 fractions by high pH reverse phase chromatography on a Dionex Ultimate 3000 LC (Thermo Scientific, Sunnyvale, CA) using an ACQUITY UPLC CSH1.7 μm C18 column (2.1 × 150 mm, Waters, Milford, MA). the same LC-MS setup was used as for the analysis of the DIA data and generation of the library. TMT MS data were analyzed in Proteome Discoverer 2.2 (Thermo Fisher Scientific, San Jose, CA) using the same databases as described above using Mascot v2.5.1 (Matrix Science) with the following settings: Enzyme was set to trypsin, with up to 1 missed cleavage. MS1 mass tolerance was set to 10 ppm and MS2 to 0.5 Da. Carbamidomethyl cysteine was set as a fixed modification and oxidation of methionine and N-terminal acetylation as variable modifications. Other modifications included the TMT-10plex modification from the quantification method (as defined in Mascot). The quantification method was set for reporter ions quantification with HCD and MS3 (mass tolerance, 20 ppm). The false discovery rate for peptide- spectrum matches (PSMs) was set to 0.01 using Percolator.41 For the reporter ion abundance only unique peptides were considered for the quantification and intensities were reported based on signal-to-noise (S/N), with no corrections applied. Protein FDR was also set to 1% in the consensus workflow. The search results were filtered by a FDR of 1% on precursor and protein level. The search results (as peptide spectrum match tables) were exported and proteins were grouped based on the IDpicker algorithm,42 which is also used in Spectronaut Pulsar X for protein grouping. Afterwards shared peptides of the UPS2 proteins with the mouse proteome were removed from the output and reporter ion channels were normalized by the median. For statistical analysis, Precursors with more than 20% missing values across the 10 channels of TMT 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/C5K14N]

[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