PXD024017

PXD024017 is an original dataset announced via ProteomeXchange.

Title	Ultra-sensitive proteomics using second generation micro pillar array LC technology with Orbitrap Exploris™ 480 and FAIMS
Description	In the light of the ongoing single-cell revolution, scientific disciplines are combining forces with the ultimate goal to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, LC separation, MS/MS data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and FAIMS. As compared to the currently commercially available pillar array columns, this dedicated limited sample column has a reduced cross section (factor of 2) and micro pillar dimensions that have been further downscaled (also by a factor of 2; 2.5 µm instead of 5 µm diameter), resulting in improved chromatography at reduced void times. A dilution series ranging from 5 to 0.05 ng/µL was prepared using trace amounts of PEG in the sample solvent to reduce adsorptive effects in the autosampler vial, sample stability up to 24h after dilution was demonstrated. Comparative processing of the MS/MS data with different database search algorithms (with and without second search feature activated and with and without rescoring based on fragmentation pattern prediction) pointed out the benefits of using Sequest HT together with INFERYS when analyzing samples at a concentration below 1 ng/µL. On average (data from quadruplicate runs), we were able to successfully identify 2855 unique protein groups from just 1 ng of HeLa lysate and this using a 60 min non-linear LC solvent gradient at a flow rate of 250 nL/min, hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10 (2436 proteins identified from 10 ng of HeLa lysate in 2019). By successfully identifying 1486 and 2210 proteins (average values, quadruplicates) from as little as 250 and 500 pg of HeLa lysate respectively, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.
HostingRepository	PRIDE
AnnounceDate	2022-02-14
AnnouncementXML	Submission_2022-02-14_01:44:10.777.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Richard Imre
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2021-02-05 03:08:23	ID requested
1	2021-06-28 07:57:06	announced
⏵ 2	2022-02-14 01:44:11	announced	2022-02-14: Updated project metadata.

Stejskal K, Op de Beeck J, D, ü, rnberger G, Jacobs P, Mechtler K, Ultrasensitive NanoLC-MS of Subnanogram Protein Samples Using Second Generation Micropillar Array LC Technology with Orbitrap Exploris 480 and FAIMS PRO. Anal Chem, 93(25):8704-8710(2021) [pubmed]

ProteomeXchange project tag: EPIC-XS

submitter keyword: micro pillar,low-input proteomics, single sell proteomics

Karl Mechtler
contact affiliation	Forschungsinstitut für Molekulare Pathologie GmbH Campus-Vienna-Biocenter 1 1030 Vienna Austria
contact email	karl.mechtler@imp.ac.at
lab head
Richard Imre
contact affiliation	IMBA Vienna
contact email	richard.imre@imba.oeaw.ac.at
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/06/PXD024017

PRIDE project URI

• PRIDE
  1. PXD024017
     1. Label: PRIDE project
     2. Name: Ultra-sensitive proteomics using second generation micro pillar array LC technology with Orbitrap Exploris™ 480 and FAIMS