Dataset Information

Evaluation of library-free strategies for phosphoproteomics and ubiquitinomics data-independent acquisition data

ABSTRACT: Phosphoproteomics and ubiquitinomics data-independent acquisition MS data is generally analyzed using a DDA spectral library. Performance of different library-free strategies of analyzing phosphoproteomics and ubiquitinomics DIA MS data are not evaluated. In this study, we assess three library-free approaches including DIA-Umpire, DIA-MSFragger and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA and diaPASEF data as well as ubiquitinomics diaPASEF data. In silico-predicted library based on DIA-NN performs best among three library-free methods, but identify less or equal phosphopeptides compared to a DDA spectral library. Furthermore, the common phosphopeptides by the predicted library and DDA library are about 50%. This case is also observed for phospho-diaPASEF data. For ubiquitinomics diaPASEF data, in silico-predicted library detects about 50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that the predicted library, although performs best in library-free methods, requires improvement for phospho DIA MS data and displays substantial advantages for ubiquitinomics diaPASEF MS data.

ORGANISM(S): Homo Sapiens

SUBMITTER: Chuanqi Zhong

PROVIDER: PXD037870 | iProX | Wed May 17 00:00:00 BST 2023

REPOSITORIES: iProX

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Publications

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

Wen Chengwen C Wu Xiurong X Lin Guanzhong G Yan Wei W Gan Guohong G Xu Xiao X Chen Xiang-Yu XY Chen Xi X Liu Xianming X Fu Guo G Zhong Chuan-Qi CQ

Journal of proteome research 20230531 7

Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted libr ...[more]

PMID: 37256709

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Project description:Data independent acquisition (DIA or DIA/SWATH ) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage,to create spectral libraries. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow window DIA methods designed to cover different slices of the precursor space, have been introduced and performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. . We found that the GPF based library outperformed library generation by direct deconvolution of the diaPASEF data, and performed comparably to deep offline fractionation libraries, when analysing diaPASEF data acquired from 200ng of commercial HeLa digest. With the ion mobility integrated GPF scheme we establish a pragmatic approach to rapid and comprehensive library generation for the analysis of diaPASEF data.

Dataset Information

Evaluation of library-free strategies for phosphoproteomics and ubiquitinomics data-independent acquisition data

Publications

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

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