PXD039576-1

PXD039576 is an original dataset announced via ProteomeXchange.

Title	Benchmarking of 5.5 cm, 50 cm and 110 cm micropillar array columns as compared to packed bed columns
Description	A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yield-ed impressive results, current studies suffer from a limited proteomic depth and dynamic range there-fore 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. Our data shows that µPAC columns clearly outperform classical packed bed columns boosting peptide IDs by up to 50% and protein IDs by up to 24%. Using the above-mentioned analysis platform, more than 10,000 proteins could be identified from a single 2 h gradient shotgun analysis for a triple proteome mix of human, yeast and E. coli digests. At high sample loads of 400 ng all three uPAC types yielded comparable number of protein identifications, whereas the 50cm neo column performed best when lower inputs of less than 200 ng were injected. Due to its unique architecture, the 5.5 cm brick column facilitates a highly meandering flow along the brick-shaped micropillars leading to an effective flow path length similar to the 50 cm neo column, while at the same time allowing high flow rates up to 2.5 µL/min. This enables to reduce overhead time by applying high flow rates during sample loading and column equilibration improving sample throughput to ~100 samples per day, while maintaining high protein ID numbers. Particularly for the single cell field, for which throughput is currently one of the most limiting factors, this column could present a valuable asset.
HostingRepository	PRIDE
AnnounceDate	2024-02-13
AnnouncementXML	Submission_2024-02-13_07:40:34.550.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Rupert Mayer
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Homo sapiens (Human); NCBI TaxID: 9606; scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2023-01-20 02:38:22	ID requested
⏵ 1	2024-02-13 07:40:35	announced

Matzinger M, Schm, ü, cker A, Yelagandula R, Stejskal K, Kr, š, š, á, kov, á G, Berger F, Mechtler K, Mayer RL, Micropillar arrays, wide window acquisition and AI-based data analysis improve comprehensiveness in multiple proteomic applications. Nat Commun, 15(1):1019(2024) [pubmed]

10.1038/s41467-024-45391-z;

submitter keyword: E. coli, Yeast, direct injection, micropillar array chromatography, Vanquish Neo, HeLa,Orbitrap Exploris 480, trap-and-elute

Karl Mechtler
contact affiliation	Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria.
contact email	karl.mechtler@imp.ac.at
lab head
Rupert Mayer
contact affiliation	Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC)
contact email	rupert.mayer@imp.ac.at
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD039576
     1. Label: PRIDE project
     2. Name: Benchmarking of 5.5 cm, 50 cm and 110 cm micropillar array columns as compared to packed bed columns