PXD005141-3

PXD005141 is an original dataset announced via ProteomeXchange.

Title	Loss-less nano-fractionator for high sensitivity, high coverage proteomics
Description	Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the online LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with mL/min-flow and relatively large column inner diameters, which allows efficient pre-fractionation but typically requires peptide amounts in the mg range. Here we describe a novel approach termed ‘spider fractionator’ in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely configurable collection mechanism, samples are concatenated in a loss-less manner into 2 to 96 fractions, with efficient peak separation. The combination of eight fractions with 100 min gradients yields very deep coverage at reasonable measurement time, and other parameters can be chosen for even more rapid or for extremely deep measurements. We demonstrate excellent sensitivity by decreasing sample amounts from 100 μg into the sub-μg range, without losses attributable to the spider fractionator and while quantifying close to 10,000 proteins. Finally, we apply the system to the rapid, automated and in-depth characterization of 12 different human cell lines to a median depth of 11,300 different proteins, which revealed differences recapitulating their developmental origin and differentiation status. The fractionation technology described here is flexible, easy to use and facilitates comprehensive proteome characterization with minimal sample requirements.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:30:42.803.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Mario Oroshi
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2016-10-13 01:40:50	ID requested
1	2017-01-30 07:53:38	announced
2	2019-08-05 02:44:04	announced	Updated contact info
⏵ 3	2024-10-22 04:30:44	announced	2024-10-22: Updated project metadata.

10.1074/mcp.O116.065136;

Kulak NA, Geyer PE, Mann M, Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics. Mol Cell Proteomics, 16(4):694-705(2017) [pubmed]

submitter keyword: Fractionation, cell lines, nano-flow

Matthias Mann
contact affiliation	Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried
contact email	mmann@biochem.mpg.de
lab head
Mario Oroshi
contact affiliation	Proteomics
contact email	oroshi@biochem.mpg.de
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/2017/04/PXD005141

PRIDE project URI

• PRIDE
  1. PXD005141
     1. Label: PRIDE project
     2. Name: Loss-less nano-fractionator for high sensitivity, high coverage proteomics