Phospho-heavy-labeled-spiketide FAIMS stepped-CV DDA (pHASED) provides real-time phosphoproteomics data to aid in cancer drug selection.

Autor: Staudt DE; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Murray HC; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Skerrett-Byrne DA; School of Environmental and Life Sciences, College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW, 2308, Australia.; Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Smith ND; Analytical and Biomolecular Research Facility (ABRF), Research Services, University of Newcastle, NSW, Callaghan, 2308, Australia., Jamaluddin MFB; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia., Kahl RGS; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia., Duchatel RJ; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Germon ZP; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., McLachlan T; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Jackson ER; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Findlay IJ; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Kearney PS; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Mannan A; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., McEwen HP; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Douglas AM; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia., Nixon B; School of Environmental and Life Sciences, College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW, 2308, Australia.; Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Verrills NM; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia., Dun MD; School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, 2308, Australia. matt.dun@newcastle.edu.au.; Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia. matt.dun@newcastle.edu.au.
Jazyk: angličtina
Zdroj: Clinical proteomics [Clin Proteomics] 2022 Dec 19; Vol. 19 (1), pp. 48. Date of Electronic Publication: 2022 Dec 19.
DOI: 10.1186/s12014-022-09385-7
Abstrakt: Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, phospho-heavy-labeled-spiketide FAIMS Stepped-CV DDA (pHASED), that employs online phosphoproteome deconvolution using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients.
(© 2022. The Author(s).)
Databáze: MEDLINE
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