Proteomics

Dataset Information

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Reproducibility, specificity and accuracy of DIA quantification - DIA analysis


ABSTRACT: For the complete description see the manuscript. Data dependent acquisition (DDA) is the method of choice for mass spectrometry based proteomics discovery experiments, data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirement to perform a DIA analysis is the availability of spectral libraries for the peptide identification and quantification. Several researches were already conducted regarding the creation of spectral libraries from DDA analyses and obtaining identifications with these in DIA measurements. But so far only few experiments were conducted, to estimate the effect of these libraries on the quantitative level. In this work we created a spike-in gold standard dataset with known contents and ratios of proteins in a complex sample matrix. With this dataset, we first created spectral libraries using different sample preparation approaches with and without sample prefractionation on peptide and protein level. Two different search engines were used for protein identification. In total, five different spike-in states were compared with DIA analyses, comparing eight different spectral libraries generated by varying approaches and one library free method, as well as one default DDA analysis. Not only the number of identifications on peptide and protein level in the spectral libraries and the corresponding analyses was inspected, but also the number of expected and identified significant quantifications and their ratios were thoroughly examined.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Myoblast, Cell Culture

SUBMITTER: Julian Uszkoreit  

LAB HEAD: Katrin Marcus

PROVIDER: PXD012988 | Pride | 2019-11-08

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
01-DirectDIA-Report.tsv Tabular
01-DirectDIA-SpectronautAnalysis.sne Other
01-DirectDIA-peptides.csv Csv
01-DirectDIA-proteins.csv Csv
01-DirectDIA.tsv Tabular
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Publications

Reproducibility, Specificity and Accuracy of Relative Quantification Using Spectral Library-based Data-independent Acquisition.

Barkovits Katalin K   Pacharra Sandra S   Pfeiffer Kathy K   Steinbach Simone S   Eisenacher Martin M   Marcus Katrin K   Uszkoreit Julian J  

Molecular & cellular proteomics : MCP 20191107 1


Currently data-dependent acquisition (DDA) is the method of choice for mass spectrometry-based proteomics discovery experiments, but data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirements to perform a DIA analysis is the availability of suitable spectral libraries for peptide identification and quantification. Several studies were performed addressing the evaluation of spectral library performance for protein identification in DIA measureme  ...[more]

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