Reproducibility, specificity and accuracy of relative quantification using spectral library-based data-independent acquisition.

Reproducibility, specificity and accuracy of relative quantification using spectral library-based data-independent acquisition. Mol Cell Proteomics. 2019 Nov 07;: Authors: Barkovits K, Pacharra S, Pfeiffer K, Steinbach S, Eisenacher M, Marcus K, Uszkoreit J Abstract 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 measurements. But so far only few experiments estimate the effect of these libraries on the quantitative level.In this work we created a gold standard spike-in sample set with known contents and ratios of proteins in a complex protein matrix that allowed a detailed comparison of DIA quantification data obtained with different spectral library approaches. We utilized in-house generated sample-specific spectral libraries created using varying sample preparation approaches and repeated DDA measurement. In addition, two different search engines were tested for protein identification from DDA data and subsequent library generation. In total, eight different spectral libraries were generated and ...
Source: Molecular and Cellular Proteomics : MCP - Category: Molecular Biology Authors: Tags: Mol Cell Proteomics Source Type: research