PXD005366-2

PXD005366 is an original dataset announced via ProteomeXchange.

Title	Robust, sensitive and automated phosphopeptide enrichment optimized for low sample amounts applied to primary hippocampal neurons
Description	Due to the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting towards an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive and less prone to variability. Here, we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on a AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive and reproducible, identifying, localizing and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications we monitored EGF-induced signaling in hippocampal neurons, starting with only 200,000 primary cells resulting in approximately 50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.
HostingRepository	PRIDE
AnnounceDate	2016-12-22
AnnouncementXML	Submission_2017-02-02_05:57:49.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Harm Post
SpeciesList	scientific name: Rattus norvegicus (Rat); NCBI TaxID: 10116; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2016-11-16 08:44:45	ID requested
1	2016-12-21 18:35:20	announced
⏵ 2	2017-02-02 05:57:50	announced	Updated publication reference for PubMed record(s): 28107008.
3	2024-10-22 04:34:32	announced	2024-10-22: Updated project metadata.

Post H, Penning R, Fitzpatrick MA, Garrigues LB, Wu W, MacGillavry HD, Hoogenraad CC, Heck AJ, Altelaar AF, Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons. J Proteome Res, 16(2):728-737(2017) [pubmed]

curator keyword: Technical, Biological

submitter keyword: Phosphoproteomics

quantification

Fe(III)-IMAC

Ti(IV)-IMAC

TiO2

BRAVO AssayMap

phosphopeptide enrichment

sensitivity

EGF

hippocampal neurons

Maarten Altelaar
contact affiliation	1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
contact email	m.altelaar@uu.nl
lab head
Harm Post
contact affiliation	Biomolecular Mass Spectrometry and Proteomics Group
contact email	h.post@uu.nl
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD005366
     1. Label: PRIDE project
     2. Name: Robust, sensitive and automated phosphopeptide enrichment optimized for low sample amounts applied to primary hippocampal neurons