Dataset Information

Wide Window Acquisition and AI-based data analysis to reach deep proteome coverage for a wide sample range, including single cell proteomic inputs – Co-immunoprecipitation samples

ABSTRACT: A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yielded impressive results, current studies suffer from a limited proteomic depth and dynamic range therefore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns (µPAC™) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYS™ search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. Using this optimized workflow on immunoprecipitation samples of Smarca5/SNF2H, we found 32 additional interaction partners compared to the original workflow utilizing a packed bed column. These additional interaction partners include previously described interaction partners of Smarca5 like Baz2b as well as undescribed interactors including Arid1a, which is also involved in chromatin remodeling and has been described as key player in neurodevelopmental and malignant disorders.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Rupert Mayer

LAB HEAD: Karl Mechtler

PROVIDER: PXD037985 | Pride | 2024-02-13

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20211105_E2_RSLC2_Schmuecker_Berger_GMI_ID520_onbead_dig_10per_control_Untagged_rep1.raw	Raw
	20211105_E2_RSLC2_Schmuecker_Berger_GMI_ID520_onbead_dig_10per_control_Untagged_rep2.raw	Raw
	20211105_E2_RSLC2_Schmuecker_Berger_GMI_ID520_onbead_dig_10per_control_Untagged_rep3.raw	Raw
	20211105_E2_RSLC2_Schmuecker_Berger_GMI_ID520_onbead_dig_10per_sample_Smarca5_Flag_rep1.raw	Raw
	20211105_E2_RSLC2_Schmuecker_Berger_GMI_ID520_onbead_dig_10per_sample_Smarca5_Flag_rep2.raw	Raw

Items per page:

1 - 5 of 51

Publications

Micropillar arrays, wide window acquisition and AI-based data analysis improve comprehensiveness in multiple proteomic applications.

Matzinger Manuel M Schmücker Anna A Yelagandula Ramesh R Stejskal Karel K Krššáková Gabriela G Berger Frédéric F Mechtler Karl K Mayer Rupert L RL

Nature communications 20240203 1

Comprehensive proteomic analysis is essential to elucidate molecular pathways and protein functions. Despite tremendous progress in proteomics, current studies still suffer from limited proteomic coverage and dynamic range. Here, we utilize micropillar array columns (µPACs) together with wide-window acquisition and the AI-based CHIMERYS search engine to achieve excellent proteomic comprehensiveness for bulk proteomics, affinity purification mass spectrometry and single cell proteomics. Our data ...[more]

PMID: 38310095

Similar Datasets

Project description:Aspergillus fumigatus can cause Invasive Pulmonary Aspergillosis (IPA), the most severe form of Aspergillus-related infections. Fungicidal azoles and fungistatic caspofungin (CAS) are the first- and second-line therapies, respectively, used to treat IPA. However, azole-resistance is on the rise and new therapeutic alternatives need to be implemented. Recently, we showed that treatment of A. fumigatus with CAS or micafungin, but not voriconazole, induces production of the oxylipin 5,8-diHODE by the fungal oxygenase PpoA. Here we investigated the influence of ppo genes, that encode fatty acid oxygenases responsible for oxylipin biosynthesis, on CAS tolerance. The ppo mutants showed a very complex pattern of CAS susceptibility. PpoA and PpoC influence on CAS tolerance is mediated by MpkA phosphorylation and protein kinase A (PKA) activity. RNAseq transcriptional profiling and label-free quantitative proteomics (spectral counts) of the ppoA, and ppoC mutants showed that differentially expressed genes and proteins are related to secondary metabolites and carbohydrate metabolism. We tested several mutants of these differentially expressed genes encoding transcription factors and signal transduction proteins and some of them are more susceptible to CAS. We also characterized two clinical isolates, CNM7555 and IFM41607, that have decreased and increased susceptibility to CAS. CNM7555 does not have increased oxylipin production in the presence of CAS while the oxylipin induction upon CAS exposure is increased in the IFM41607, suggesting oxylipins are not the single mechanism involved in CAS tolerance in these isolates. Upon CAS exposure, CNM7555 has increased MpkA phosphorylation and PKA activity than IFM41607. Modulation of secondary metabolite production and carbohydrate metabolism is also essential in these clinical isolates for acquiring CAS tolerance. Our results reveal different aspects and genetic determinants involved in A. fumigatus CAS tolerance.