PXD082116-1

PXD082116 is an original dataset announced via ProteomeXchange.

Title	Mass spectrometry to identify RNA binding proteins interacting with PACERR RNA
Description	Protein lysates NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific 78833) was used to isolate nuclear proteins from THP-1 macrophages. In vitro transcribed RNA PACERR cloned into pGEM-7Z+ (Promega P2251) was the template to in vitro-transcribe RNA using T7 RNA polymerase, and biotin-16-UTP was incorporated at approximately every 20th to 25th nucleotide of the transcript. An antisense PACERR RNA sequence and GFP sequence (SP6) were used as controls for specificity of the in vitro transcribed product. GFP is approximately the same size as the human PACERR transcript, 715bp versus 825bp. RNA/Protein pulldown Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific 20164) was used to identify proteins bound to PACERR IVT RNA. Biotinylated RNA was incubated with nuclear cellular extracts from THP-1 macrophages unstimulated or treated with 50µg/ml HDL. Streptavidin magnetic beads were used to pull-down biotinylated PACERR. Proteins bound to PACERR were identified using liquid chromatography-mass spectrometry (LC-MS). Protein concentrations were measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific 23225) Protein trypsin digestion and first dimension RPLC Pull-down protein solutions were digested by the FASP procedure. The protein samples were supplemented with 50 mM Tris-HCl buffer with 1M Urea (pH 8.0) to a final volume of 93.5 μL. Protein samples were incubated with 5 mM DTT (final concentration) for 30 min at 56 ℃, then added iodoacetamide (IAA) to a 14.85 mM final concentration, and incubated in the dark at room temperature for 15 min. After these procedures, protein samples were loaded into 10 kD Microcon filtration devices (Millipore) and centrifuged at 12,000 × g for 20 min and washed three times with 50 mM Tis-HCl buffer. Then the samples were digested using trypsin/Lys-C mix (Promega) at an enzyme to protein mass ratio of 1:25 overnight at 37 °C. Peptides were extracted and dried (SpeedVac, Eppendorf). The digested peptides were then desalted by reverse phase stage tips
HostingRepository	PRIDE
AnnounceDate	2026-08-13
AnnouncementXML	Submission_2026-08-13_04:06:22.468.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Robin Joshi
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Xevo G2-XS QTof

Revision	Datetime	Status	ChangeLog Entry
0	2026-08-03 19:32:59	ID requested
⏵ 1	2026-08-13 04:06:23	announced

Dataset with its publication pending

submitter keyword: mass spectrometery,Proteomics, PACERR

Garret A. FitzGerald
contact affiliation	University of Pennsylvania
contact email	garret@upenn.edu
lab head
Robin Joshi
contact affiliation	University of Pennsylvania
contact email	robin.joshi@pennmedicine.upenn.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD082116
     1. Label: PRIDE project
     2. Name: Mass spectrometry to identify RNA binding proteins interacting with PACERR RNA