PXD045405

PXD045405 is an original dataset announced via ProteomeXchange.

Title	Heat ‘n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour
Description	Clinical proteomics on solid tissue samples from human tumour biopsies present multiple challenges. These include limited sample availability, reducing endogenous protease activity and challenges with efficiently solubilising and digesting proteins for high throughput mass spectrometry while achieving comprehensive proteome coverage. Protocols for cancer biopsy proteomics involves three sample preparation steps: tissue washing, lysis and digestion, followed by peptide clean-up by SPE. Our team have reported improvements in all three steps that greatly reduce processing time, enabling greater automation and higher sample throughput. Our aim was to develop a universal protocol for tissue sample preparation that is quick and simple while improving protein solubilization, proteome coverage and reproducibility. Sample preparation with small (<2 mg) biopsies pose additional limitations including the risk of sample loss and introducing variability with multiple reagent additions steps. We now report a one-pot method called heat and beat (HnB). By identifying the limitations and challenges of the existing protocols, we were able to develop a new protocol with fewer steps by achieving lysis, reduction, alkylation and digestion in essentially a single step which was preceded by a 7 min sample heat treatment step. This was achieved by several changes to the existing protocol including the combined use of a beadbeater for tissue homogenisation followed by pressure cycling technology (PCT) using a Barocycler to further lyse and digest the sample. The procedure requires 38 minutes and was tested on 6 tissue types, across three tissue embedding techniques, fresh frozen, OCT embedded (FF-OCT), and FFPE. Our results show that HnB is an efficient new one-pot procedure. The protocol significantly reduced endogenous protease activity, improves peptide yield by up to 3-fold and delivers digestion efficiency of 85-90% from each sample source. We obtained an increase in the number of proteins identified of up to two-fold while reducing variability and processing time.
HostingRepository	PRIDE
AnnounceDate	2025-03-26
AnnouncementXML	Submission_2025-03-26_00:58:50.299.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dylan Xavier
SpeciesList	scientific name: Rattus norvegicus (Rat); NCBI TaxID: 10116; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	iodoacetamide derivatized residue
Instrument	TripleTOF 6600

Revision	Datetime	Status	ChangeLog Entry
0	2023-09-14 22:39:58	ID requested
⏵ 1	2025-03-26 00:58:50	announced

10.1021/ACS.ANALCHEM.3C04708;

submitter keyword: sample preperation,proteomics, high-throughput, Heat n Beat, universal,mass spec

Phillip J. Robinson
contact affiliation	ProCan®, Children’s Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia.
contact email	probinson@cmri.org.au
lab head
Dylan Xavier
contact affiliation	Children's Medical Research Institute Procan
contact email	dxavier@cmri.org.au
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/2025/03/PXD045405

PRIDE project URI

• PRIDE
  1. PXD045405
     1. Label: PRIDE project
     2. Name: Heat ‘n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour