Dataset Information

Plasma Proteomics Benchmarking and Optimisation/ Plasma proteomics across three generations of mass spectrometry instruments: lessons for optimising biomarker discovery

ABSTRACT: In biomarker discovery phases clinical proteomics provides large-scale identification and quantification of proteins in donor samples. For these studies plasma is the most frequently used biofluid, as it reflects both physiological and pathological states of the circulating proteome. However, the vast dynamic range of proteins in plasma remains a significant challenge, where low-abundance biomarkers are suppressed by high-abundance proteins, despite substantial technological improvements. Often the focus of improving outcomes in plasma proteomic workflows are within the biofluid’s sample preparation, although adjusting MS methods dramatically improves detection and sampling of lower abundance proteins. Herein we have benchmarked various liquid chromatography (LC) and data-independent acquisition (DIA) methods across three generations of mass spectrometry instruments: timsTOF Pro (2017), Orbitrap Eclipse (2020), and Orbitrap Astral (2023). This study explored 27 methods across 200 hours of instrument acquisition time, encompassing varying LC, MS, and database searching workflows. While each instrument generation significantly improved performance, each instrument also revealed a unique and different tuneable range to improve performance in plasma samples, highlighting the benefit of investing in plasma-specific method development for any mass spectrometer. We also evaluated the detection and quantification capabilities of each instrument via a unique approach of mixing paired Platelet Poor Plasma (PPP) and Platelet Rich Plasma (PRP), introducing linear contamination markers for quantitative assessment. This approach tested each instrument's sensitivity to detect low-abundance peptides and evaluated their quantitative accuracy. Lastly, we performed testing of the Orbitrap Astral’s parallel ion processing capabilities with the aim of improving low-abundance protein identifications using gas-phase enrichment (GPE). More specifically, we show that optimisation of MS2 AGC targets and injection time enhanced GPE of low-abundance peptides, improving detection in plasma samples.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Jack Edwards

LAB HEAD: Laura Dagley

PROVIDER: PXD069060 | Pride | 2026-04-21

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 384

Publications

Plasma Proteomics Across Three Generations of Mass Spectrometry Instruments: Lessons for Biofluid Method Optimisation.

Edwards Jack J Multari Dylan H DH Dite Toby T Binos Steve S Shugg Clare C Webb Andrew I AI Dagley Laura F LF Emery-Corbin Samantha J SJ

Proteomics 20260415

In biomarker discovery phases clinical proteomics provides large-scale identification and quantification of proteins in donor samples. For these studies plasma is the most frequently used biofluid, as it reflects both physiological and pathological states of the circulating proteome. However, the vast dynamic range of proteins in plasma remains a significant challenge, where low-abundance biomarkers are suppressed by high-abundance proteins, despite substantial technological improvements. Often ...[more]

PMID: 41987532

Similar Datasets

Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA. Cell culture conditions and media Human fibroblast cell strains (WS: AG05229, AG12795, AG12797; BS: GM02932, GM03402, GM16891; RTS: AG18371, AG18375, AG05013; Normal/Wild-type: AG04054, AG06310, AG09975) were obtained from the Coriell Repository (Camden, NJ), from donors matched for gender and of similar ages, and were at similar passage levels. Cells were cultured in MEM supplemented with Earle’s salts, 20% fetal bovine serum, 1x penicillin/streptomycin, and 1x fungizone in 3% O2 at 37oC and harvested for RNA extraction during active growth and at ~ 80% confluence. GeneChip microarray expression Total RNA from the 12 fibroblast cell strains was isolated by extraction with TRIzol (Invitrogen) and purified using the RNeasy system (Qiagen). Total RNA was amplified by in vitro transcription using the Ovation RNA Amplification System V2 (NuGen). The resultant cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen), and then purified using QIAquick columns (Qiagen), as specified by the Ovation System manual. Labeled probe was hybridized to Affymetrix U133A 2.0 GeneChips, and ultimately scanned using an Axon GenePix array scanner. Statistical analysis of microarray expression experiment The output files were normalized by Robust Multiarray Average (RMA), using the R package GCRMA and gene expression levels were log2-transformed.

			Action	DRS
	P4776_101_15_1_R1.raw	Raw
	P4776_101_15_1_R2.raw	Raw
	P4776_101_15_1_R3.raw	Raw
	P4776_101_15_2_R1.raw	Raw
	P4776_101_15_2_R2.raw	Raw