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CysQuant: simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

ABSTRACT: Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function, urging the need for accurate cysteine oxidation quantification to decode protein redox regulation. Here, we present a novel approach called CysQuant that enables simultaneous quantification of cysteine oxidation degrees and protein abundancies. Reduced and reversibly oxidized cysteines are differentially labeled with light and heavy iodoacetamide isotopologues and analyzed using data-dependent acquisition (DDA) or data-independent acquisition (DIA) mass spectrometry. Using in silico predicted spectral libraries, plexDIA quantified on average 18% oxidized in the model plant Arabidopsis thaliana, though revealed a subset of highly oxidized cysteines part of disulfide bridges in AlphaFold2 predicted protein structures. Studying protein redox regulation of plant seedlings in response to excessive light, CysQuant successfully identified the well-established increased reduction of Calvin-Benson cycle enzymes, in addition to discovery of other, yet uncharacterized redox-sensitive disulfides in plastidial enzymes. CysQuant is widely applicable to diverse mass spectrometry platforms studying the cysteine modification status.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Shoot

SUBMITTER: Patrick Willems

LAB HEAD: Kris Gevaert

PROVIDER: PXD043895 | Pride | 2023-07-21

REPOSITORIES: Pride

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			Action	DRS
	B2430_Bp_IonOpt_CMB-1251_KRGEV_Willems_1_DDA_20230412150004.raw	Raw
	B2432_Bp_IonOpt_CMB-1251_KRGEV_Willems_1_DIA.raw	Raw
	B2432_Bp_IonOpt_CMB-1251_KRGEV_Willems_1_DIA.raw.dia	Raw
	B2432_Bp_IonOpt_CMB-1251_KRGEV_Willems_1_DIA.raw.dia.quant	Raw
	B2434_Bp_IonOpt_CMB-1251_KRGEV_Willems_5_DDA.raw	Raw

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Project description:Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation changes without considering the variation in the total levels of the proteins. However, the regulation of protein synthesis and protein degradation are also associated with many of the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in the Post Translational Modifications (PTMs) focused analyses, such as cysteine redox characterization. This aspect was only address in the cysTMTRAQ method, which combines two types of isobaric tags in the same experiment leading to a considerable increase in the costs of the analysis. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using non-isotopically labeled alkylating reagents (oxSWATH) is presented, thus integrating the information regarding relative cysteine oxidation with the comparative analysis of the total protein levels in a cost effective highthrouput approach. The proposed method was tested using a redox regulated protein, and further applied to a comparative analysis of secretomes obtained under control or oxidative stress conditions to strengthen the importance of considering the changes in protein total levels. OxSWATH allowed to determine the relative proportion of reduced and reversible oxidized oxoforms of the proteins, and by considering total protein levels, to determine the total levels of each fraction, which are then used for comparative analysis. This approach can be an important tool in redox centered approaches, being able to not only analyze the overall reduce/reversible oxidized state of proteins but able to be further applied in the characterization of the different oxoforms of the individual cysteines. Moreover, since samples are acquired in SWATH-MS mode, besides the redox centered analysis, a generic differential protein expression analysis can be also performed, allowing a truly comprehensive evaluation of proteomics changes upon oxidative stimulus.

Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed âantioxidant responseâ or âPhase II detoxificationâ. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental âimageâ of UV generated lipid mediators. Experiment Overall Design: we profiled global mRNA expression levels in human dermal fibroblasts that had been treated with either UVA-1 or oxidized lipids. To investigate the effect of oxidized phospholipids on gene regulation, we used two preparations, which differed in their degree of oxidation; the minimally oxidized UV-PAPC resulting from UVA-1 irradiation of PAPC, and air-oxidized PAPC (OxPAPC), which represents the full spectrum of oxidation products (Gruber 07) (Reis et al., 2005). We irradiated dermal fibroblasts with UVA-1 (40J/cmÂ²) or treated them with UV-PAPC, OxPAPC or native PAPC (100Âµg/ml each). We analyzed global gene expression four hours after stimulation with gene arrays (Affymetrix U133A Plus 2.0 Gene Chips).

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CysQuant: simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

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