PXD070352

PXD070352 is an original dataset announced via ProteomeXchange.

Title	UltraPlex: Expanding isobaric hyperplexing via orthogonal protease cleavage
Description	Sample preparation was conducted using an adaptation of our SimPLIT workflow10. Four cell pellets corresponding to four different colorectal cancer cell lines were lysed in a buffer containing 1% sodium deoxycholate (SDC, Merck, Cat. No. 30970), 100 mM triethylammonium bicarbonate (TEAB, Merck, Cat. No. T7408), 10% isopropanol and 50 mM NaCl, freshly supplemented with 5 mM TCEP (Thermo, Bond-breaker Cat. No 77720), 10 mM iodoacetamide (IAA, Merck, Cat. No. I1149), universal nuclease 1:2000 vol/vol (Pierce, Cat. No. 88700) and Halt protease and phosphatase inhibitor cocktail (Thermo, Cat. No. 78442, 100X) with 5 min of bath sonication. Protein concentration was measured with the Quick Start Bradford protein assay (Bio-Rad, Cat. No. 5000205). Aliquots of 30 μg of total protein from each cell line were transferred in a 96-well plate according to the 58-plex design. An E. coli protein extract (Bio-Rad, Cat. No. 1632110) was added at 1 μg or 0.5 μg across the human cell lysates. The plate was dried and the samples for the arginine-specific cleavage set (TrypR) were reconstituted in 25 μL (TMT11 subset) or 12.5 μL (TMT18 subset) of 100 mM TEAB buffer followed by the addition of 10 μL TMT11 (stock 20 μg/μL, Thermo Fischer Scientific, Cat. No. A34808) or 5 μL TMTpro18 (stock 25 μg/μL, Thermo Fischer Scientific, Cat. No. A52045), and 1h reaction at room temperature for protein-level TMT labelling. The TMT labelled samples were SpeedVac dried, and all samples (labelled and unlabelled) were reconstituted in 20 μL of 100 mM TEAB buffer. Proteins were digested overnight at room temperature by the addition of 2 μL LysC (Thermo Fischer Scientific, Cat. No. 90307) or Trypsin (Thermo Fischer Scientific, Cat. No. 90059) stock solution of 500 ng/μL in 0.1% formic acid (final enzyme 50 ng/μL, 1:30) for the LysC and TrypR sets respectively. The peptides were then fully labelled by another addition of 10 μL TMT11 or 5 μL TMTpro18. The reaction was quenched after 1h with the addition of 2 μL 5% hydroxylamine solution and premix samples were prepared by pooling 0.5 μL from each sample into 100 μL of 0.1% TFA for each sub-plex separately. In each premix, SDC was precipitated with addition of formic acid at 2% and centrifugation at 20,000 × g for 5 minutes. Supernatants were transferred into clean vials for LC-MS analysis. Following evaluation of the premix runs, samples were pooled into four sub-plexes, diluted with 100 μL of HPLC water, heated at 95 °C for 5 min, cooled down and heated for another 5 minutes. Lastly, each pool was acidified with formic acid at 2%, the precipitated SDC was removed by centrifugation and supernatants were SpeedVac dried. High-pH Reversed-phase Peptide Fractionation All four sub-plexes were pooled in 100 μL of 0.1% (v/v) ammonium hydroxide and the final peptide pool was fractionated with high pH Reversed-Phase chromatography using the XBridge C18 column (2.1 × 150 mm, 3.5 μm, Waters) on an UltiMate 3000 HPLC system over a 1% gradient in 35 min. Mobile phase A was 0.1% (v/v) ammonium hydroxide and mobile phase B was 0.1% ammonium hydroxide (v/v) in acetonitrile. Fractions were collected every 30 sec and eventually pooled into 45 samples for LC-MS analysis.
HostingRepository	PRIDE
AnnounceDate	2026-01-30
AnnouncementXML	Submission_2026-01-30_14:19:38.943.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Graeme Benstead-Hume
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-11-05 04:16:06	ID requested
⏵ 1	2026-01-30 14:19:39	announced

Dataset with its publication pending

submitter keyword: None

Jyoti Choudhary
contact affiliation	ICR, Functional Proteomics
contact email	jyoti.choudhary@icr.ac.uk
lab head
Graeme Benstead-Hume
contact affiliation	ICR
contact email	ghume@icr.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD070352
     1. Label: PRIDE project
     2. Name: UltraPlex: Expanding isobaric hyperplexing via orthogonal protease cleavage