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A hybrid DDA/DIA-PASEF based assay library for a deep proteotyping of triple-negative breast cancer
Petr Lapcik1, Klara Synkova1, Lucia Janacova1
1Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic.
Abstract:
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and deeper proteome coverage is needed for its molecular characterization. We present comprehensive library of targeted mass spectrometry assays specific for TNBC and demonstrate its applicability. Proteins were extracted from 105 TNBC tissues and digested. Aliquots were pooled, fractionated using hydrophilic chromatography and analyzed by LC-MS/MS in data-dependent acquisition (DDA) parallel accumulation-serial fragmentation (PASEF) mode on timsTOF Pro LC-MS system. 16 individual lysates were analyzed in data-independent acquisition (DIA)-PASEF mode. Hybrid library was generated in Spectronaut software and covers 244,464 precursors, 168,006 peptides and 11,564 protein groups (FDR = 1%). Application of our library for pilot quantitative analysis of 16 tissues increased identification numbers in Spectronaut 18.5 and DIA-NN 1.8.1 software compared to library-free setting, with Spectronaut achieving the best results represented by 190,310 precursors, 140,566 peptides, and 10,463 protein groups. In conclusion, we introduce assay library that offers the deepest coverage of TNBC proteome to date. The TNBC library is available via PRIDE repository (PXD047793).
Insights
Researchers developed a comprehensive mass spectrometry assay library for triple-negative breast cancer (TNBC), enabling deeper proteome coverage for this aggressive cancer subtype. This new TNBC proteome resource enhances protein identification in molecular characterization studies.
Area of Science:
- Proteomics
- Cancer Research
- Mass Spectrometry
Background:
- Triple-negative breast cancer (TNBC) is an aggressive subtype requiring advanced molecular characterization.
- Existing proteomic coverage for TNBC is insufficient for comprehensive analysis.
Purpose of the Study:
- To develop and validate a comprehensive targeted mass spectrometry assay library specific for TNBC.
- To enhance the depth of proteome coverage for TNBC molecular studies.
Main Methods:
- Proteins extracted from 105 TNBC tissues and analyzed using LC-MS/MS with data-dependent acquisition (DDA)-PASEF.
- A hybrid library was generated using Spectronaut software, covering 244,464 precursors and 11,564 protein groups.
- Pilot quantitative analysis of 16 tissues was performed using the developed library in Spectronaut and DIA-NN software.
Main Results:
- The TNBC assay library provides the deepest proteome coverage to date for this cancer subtype.
- Application of the library significantly increased protein identification numbers compared to library-free methods.
- Spectronaut software achieved superior results, identifying 190,310 precursors and 10,463 protein groups.
Conclusions:
- A novel assay library offers unprecedented proteome coverage for triple-negative breast cancer.
- This resource facilitates improved molecular characterization and quantitative analysis of TNBC.
- The TNBC library is publicly available via the PRIDE repository (PXD047793).
