PXD041387-2

PXD041387 is an original dataset announced via ProteomeXchange.

Title	SLAPSHOT reveals rapid dynamics of extracellularly exposed proteome in response to calcium-activated plasma membrane phospholipid scrambling
Description	To facilitate our understanding of the often rapid and nuanced dynamics of extracellularly exposed proteomes during signaling events, it is important to devise robust workflows affording fast time resolution without biases and confounding factors. Here, we present Surface-exposed protein Labeling using PeroxidaSe, H2O2, and Tyramide-derivative (SLAPSHOT), to label extracellularly exposed proteins in a rapid, sensitive, and specific manner, while preserving cellular integrity. This experimentally simple and flexible method utilizes recombinant soluble APEX2 peroxidase that is applied to cells, thus circumventing biological perturbations, tedious engineering of tools and cells, and labeling biases. APEX2 neither requires metal cations for activity nor contains disulfide bonds, conferring versatility for a wide spectrum of experimental setups. We applied SLAPSHOT followed by quantitative mass spectrometry-based proteomics analysis to examine the immediate and extensive cell surface expansion and ensuing restorative membrane shedding upon the activation of Scott syndrome-linked TMEM16F, a ubiquitously expressed calcium-dependent phospholipid scramblase and ion channel. Time-course data ranging from one to thirty minutes of calcium stimulation using wild-type and TMEM16F deficient cells revealed intricate co-regulation of known protein families, including those in the integrin and ICAM families. Crucially, we identified proteins that are known to reside in intracellular organelles, including ER, as occupants of the freshly deposited membrane, and mitovesicles as an abundant component and contributor to the extracellularly exposed proteome. Our study not only provides the first accounts of the immediate consequences of calcium signaling on the extracellularly exposed proteome, but also presents a blueprint for the application of SLAPSHOT as a general approach for monitoring extracellularly exposed protein dynamics.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:57:02.916.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Sami Tuomivaara
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	acetylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2023-04-06 12:14:19	ID requested
1	2024-08-29 10:36:59	announced
⏵ 2	2024-10-22 06:57:03	announced	2024-10-22: Updated project metadata.

10.1038/S42003-024-06729-X;

submitter keyword: Calcium signaling, Surface proteomics,LC-MSMS, Isobaric labeling, Phospholipid scrambling

Arun Paul Wiita
contact affiliation	Department of Laboratory Medicine University of California, San Francisco USA
contact email	arun.wiita@ucsf.edu
lab head
Sami Tuomivaara
contact affiliation	University of California, San Francisco
contact email	sami.tuomivaara@ucsf.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD041387
     1. Label: PRIDE project
     2. Name: SLAPSHOT reveals rapid dynamics of extracellularly exposed proteome in response to calcium-activated plasma membrane phospholipid scrambling