信息摘要：
2026年，上海交通大学医学院团队在Research Square平台发表预印本论文《SIRT2 functions as an N-acetylaspartate hydrolase that counteracts cardiac metabolic remodeling in kidney disease》(DOI: 10.21203/rs.3.rs-8510072/v1)，研究中使用LUYOR UCL-3200L紫外交联仪对光亲和标记的N-乙酰天冬氨酸(NAA)探针进行365 nm紫外辐照交联，用于NAA互作蛋白的鉴定，为揭示SIRT2作为NAA水解酶、NAA经竞争性抑制MDH1扰乱心肌代谢重塑的机制研究提供关键实验证据。
上海交通大学医学院将LUYOR UCL-3200L用于NAA互作蛋白交联实验
2026年，上海交通大学医学院团队在Research Square平台发表预印本论文《SIRT2 functions as an N-acetylaspartate hydrolase that counteracts cardiac metabolic remodeling in kidney disease》，研究中使用LUYOR UCL-3200L紫外交联仪用于NAA互作蛋白交联实验，对与光亲和标记NAA探针孵育后的小鼠心脏组织裂解液进行365 nm紫外辐照交联，使探针标记的NAA互作蛋白共价交联固定，用于富集并鉴定NAA的直接作用靶点。UCL-3200L紫外交联仪在基于活性的蛋白质谱分析(ABPP)流程中承担关键的交联固定环节，通过365 nm紫外辐照使光亲和探针与互作蛋白稳定交联，确保后续点击化学偶联、链霉亲和素磁珠富集及质谱鉴定等步骤中靶蛋白不丢失，UCL-3200L紫外交联仪为NAA靶蛋白鉴定结果的可重复性与可靠性提供保障。
上海交通大学医学院位于上海，是集医疗、教学、科研于一体的大型综合性医学院校。该研究团队围绕慢性肾脏病(CKD)相关心肌重塑的发病机制开展研究，利用靶向代谢组学锁定循环N-乙酰天冬氨酸(NAA)为与早期心功能障碍关联最密切的代谢物，并通过基于活性的蛋白质谱分析(ABPP)鉴定出胞质苹果酸脱氢酶1(MDH1)与去乙酰化酶SIRT2为NAA的直接靶点，系统揭示了NAA竞争性抑制MDH1、破坏苹果酸-天冬氨酸穿梭与心肌能量代谢，以及SIRT2作为NAA水解酶缓解NAA代谢压力的SIRT2-NAA-MDH1轴机制。UCL-3200L紫外交联仪用于的NAA互作蛋白交联实验作为鉴定NAA直接靶点的关键手段，为上述分子机制的确认提供了重要实验证据。
本实验采用LUYOR UCL-3200L作为NAA互作蛋白交联实验的核心交联设备，用于对与光亲和标记NAA探针孵育后的蛋白样品进行365 nm紫外辐照交联。实验将光亲和标记的NAA探针(Cat. No.: 1132432，WuXi AppTec)与新鲜制备的小鼠心脏组织裂解液于4℃孵育2 h，经UCL-3200L紫外交联仪365 nm紫外辐照10 min，使探针与NAA互作蛋白共价交联；随后加入TCEP、TBTA、CuSO4与Biotin-azide进行点击化学偶联，经链霉亲和素磁珠富集、SDS-PAGE分离与考马斯亮蓝染色后，切取蛋白条带进行质谱鉴定。UCL-3200L紫外交联仪用于NAA互作蛋白交联结果表明，NAA探针组特异性富集到56个候选蛋白，蛋白按肽谱匹配数(PSMs)和覆盖度排序，包含MDH1、SIRT2、SIRT3等，为后续证实NAA竞争性抑制MDH1、SIRT2作为NAA水解酶提供了直接实验证据。
该研究证实，LUYOR UCL-3200L紫外交联仪可用于光亲和标记-点击化学联用的互作蛋白鉴定流程中蛋白样品的365 nm紫外交联固定，适用于代谢物-蛋白互作鉴定、靶点垂钓(ABPP)等分子生物学应用场景。LUYOR UCL-3200L紫外交联仪在国内外高校和科研院所得到广泛好评，欢迎私信或留言上海路阳了解详情！
原文段落
NAA互作蛋白鉴定实验方法原文
NAA-binding proteins were identified as previously described55,56. Heart tissues were harvested from adult mice and immediately washed with ice-cold phosphate buffer. Apex tissues (50 mg) were minced and lysed in 500 μL ice-cold 0.5% NP-40 buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin and 1 mM PMSF) using a tissue grinder. Lysates were clarified by centrifugation at 15,000 × g for 20 min at 4 °C. The supernatants were incubated with 100 μM synthesized NAA probes (1132432, AppTec) or NAA at 4 °C for 2 h, followed by exposure to 365 nm UV light (UCL-3200L, LUYOR) for 10 min to crosslink NAA-interacting proteins. Subsequently, the reaction mixtures were adjusted to final concentrations of 1 mM TCEP (C4706, Sigma-Aldrich), 0.1 mM TBTA (678937, Sigma-Aldrich), 1 mM CuSO4 (C1297, Sigma-Aldrich), and 1 mM Biotin-azide (HY-129832, MedChemExpress), and incubated at 4 °C for 1 h for click chemistry. Protein aggregates were removed by centrifugation at 20,000 × g for 15 min, and the supernatants were incubated with streptavidin magnetic beads (HY-K0208, MedChemExpress) for 2 h with gentle rotation at 4 °C. Beads were washed three times with 700 μL 0.5% NP-40 buffer and then resuspended in 100 μL SDS-PAGE loading buffer. Samples were resolved by SDS-PAGE, stained with Coomassie Blue Fast Staining Solution (P0017, Beyotime), and protein bands were excised for mass spectrometry analysis.
NAA互作蛋白鉴定结果原文
To elucidate the molecular mechanism by which NAA contributes to cardiac dysfunction, we employed an activity-based protein profiling (ABPP) approach using NAA probes as bait. Photoaffinity-tagged NAA probes were synthesized to enable enrichment and visualisation of interacting proteins (Extended Data Fig. 4a). The pro-hypertrophic effects of probe-tagged NAA in cardiomyocytes were comparable to those of non-tagged NAA (Extended Data Fig. 4b), validating the functional integrity of probe-tagged NAA. The NAA probes were incubated with freshly prepared heart lysates for target labelling, and an equivalent concentration of non-tagged NAA was used as a control (Extended Data Fig. 4c). In the NAA probe group, 56 candidate proteins were specifically enriched (Extended Data Fig. 4d). The top 10 proteins were ranked according to their peptide-spectrum matches (PSMs) and coverage (Fig. 2a).
研究结论原文
These findings expand sirtuins from protein deacylases to direct regulators of small-molecule metabolites and establish NAA as a kidney–heart metabolic mediator, revealing an amino acid acetylation-dependent layer of energy homeostasis.
DOI:doi.org/10.21203/rs.3.rs-8510072/v1
