PXD043678

PXD043678 is an original dataset announced via ProteomeXchange.

Title	A magnetic bead-based workflow for sensitive and streamlined cell surface proteomics
Description	Cell-surface proteins represent an important class of molecules for therapeutic targeting and defining cellular phenotypes. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, posttranslational modifications, hydrophobic regions, and processing requirements. To improve the identification of cell-surface proteins via their corresponding N-linked glycopeptides (N-glycopeptides), we optimized a Cell-Surface Capture (CSC) workflow which incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), non-enzymatic deamidation (digestion and de-glycosylation buffers), and data acquisition methods. Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid and streptavidin magnetic beads for maximal N-glycopeptide detection. Furthermore, single-pot solid-phased-enhanced sample-preparation (SP3) circumvented the need to isolate cell membranes by affording the use of strong detergents and chaotropes for protein extraction. Notably, with semi-automated processing, sample handling was simplified and between ~600-900 N-glycoproteins were identified from only 25-200µg of HeLa protein. Overall, the improved efficiency of the magnetic-based CSC workflow allowed us to identify both previously reported and novel N-glycosites with less material and high reproducibility, and should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.
HostingRepository	PRIDE
AnnounceDate	2024-01-21
AnnouncementXML	Submission_2024-01-20_18:26:26.734.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dylan Dieters-Castator
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	deamidated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Eclipse; Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2023-07-11 09:38:25	ID requested
⏵ 1	2024-01-20 18:26:27	announced

10.1021/acs.jproteome.3c00432;

Dieters-Castator DZ, Manzanillo P, Yang HY, Modak RV, Rardin MJ, Gibson BW, Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics. J Proteome Res, 23(2):618-632(2024) [pubmed]

submitter keyword: LC-MS

Mass Spectrometry

Surfaceomics

Cell Surface Capture

N-glycopeptide Enrichment

Plasma Membrane N-glycoprotein

Matt Rardin
contact affiliation	Amgen
contact email	mrardin@amgen.com
lab head
Dylan Dieters-Castator
contact affiliation	Covant Therapeutics
contact email	ddieters@gmail.com
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/2024/01/PXD043678

PRIDE project URI

• PRIDE
  1. PXD043678
     1. Label: PRIDE project
     2. Name: A magnetic bead-based workflow for sensitive and streamlined cell surface proteomics