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Multiplexed phosphoproteomics of low cell numbers using SPARCE

ABSTRACT: Understanding cellular diversity and disease mechanisms requires global analysis of proteins and their modifications. Ultrasensitive mass spectrometry-based proteomics enables unbiased protein-level analysis of low cell numbers, down to single cells. However, phosphoproteomics remains limited to high-input samples due to sample losses and poor reaction efficiencies associated with processing low cell numbers. In this study, we have used isobaric stable isotope labelling as a promising approach for reproducible and accurate quantification of low abundant phosphopeptides. Here, we introduce SPARCE (Streamlined Phosphoproteomic Analysis of Rare CElls) for multiplexed phosphoproteomic analysis of low cell numbers. SPARCE outperforms traditional methods by enhancing labelling efficiency and phosphoproteome coverage. To demonstrate the utility of SPARCE, we analysed four patient-derived glioblastoma stem cell lines, reliably quantifying phosphosite changes from 1,000 FACS-sorted cells. This workflow expands the possibilities for signalling analysis of rare cell populations.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Cell Culture

DISEASE(S): Brain Cancer

SUBMITTER: Silvia Surinova

LAB HEAD: Silvia Surinova

PROVIDER: PXD056902 | Pride | 2025-04-01

REPOSITORIES: Pride

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Project description:Background: Ischemic preconditioning (IPC), i.e., brief periods of ischemia, protect the heart from subsequent prolonged ischemic injury, and reduces infarction size. Myocardial stunning refers to transient loss of contractility in the heart after myocardial ischemia that recovers without causing permanent damage. The relationship between IPC and myocardial stunning remains incompletely understood. Purpose: The primary aim of this study was to examine the effects of IPC on the relationship between ischemia duration, stunning, and infarct size in an ischemia-reperfusion injury model. The secondary aim of the study was to examine to which extent the phosphoproteomic changes induced by IPC relate to myocardial contractile function. Methods: Rats were subjected to different durations of left anterior descending artery (LAD) occlusion, with or without preceding IPC. Echocardiograms were acquired at 4 and 48 hours to assess cardiac contraction in the affected myocardial segment. Reversible akinesia was defined as the presence of myocardial akinesia at 4 hours that resolved by 48 hours; and was considered to represent myocardial stunning. Infarction size was evaluated using triphenyl tetrazolium chloride staining. Phosphoproteomic analysis was performed in heart tissue from preconditioned and non-preconditioned animals using nano-liquid chromatography-mass spectrometry. Results: Reversible akinesia was observed in a majority of the rats that were subjected to IPC and subsequently exposed to ischemia of 13.5 or 15 minutes of ischemia (83.3% [n/N] and 66.6% [n/N] respectively). Among rats without IPC, who were exposed to either 10, 11, 12 or 13.5 minutes of ischemia, reversible akinesia was observed in 0% (n/N), 17% (2/12), 0% (n/N) and 0% (n/N) of rats (p<0.001). Phosphoproteomic analysis revealed significant differential regulation of 809 phosphopeptides between IPC and non-IPC groups, with significant associations with the sarcomere, Z-disc, and actin binding. Conclusion: Our study shows that IPC preferentially induces changes in phosphosites of proteins involved in myocardial contraction, and both increases the incidence of reversible post-ischemic myocardial stunning after ischemia-reperfusion injury and reduces infarction size.

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Multiplexed phosphoproteomics of low cell numbers using SPARCE

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