<br>完整 19S、20S、26S 和 30S 蛋白酶体的 CDMS 分析：低 pH 下高阶 20S 组装的证据†,Analytical Chemistry

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完整 19S、20S、26S 和 30S 蛋白酶体的 CDMS 分析：低 pH 下高阶 20S 组装的证据†
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-08-08 , DOI: 10.1021/acs.analchem.3c00472
Adam J Anthony ₁ , Amit K S Gautam ₂ , Lohra M Miller ₁ , Yiran Ma ₁ , Anya G Hardwick ₁ , Anu Sharma ₃ , Subhadip Ghatak ₃ , Andreas Matouschek ₂ , Martin F Jarrold ₁ , David E Clemmer ₁

Affiliation

电荷检测质谱（CDMS）被作为研究蛋白酶体的一种手段进行了检验。为此，测量了来自酿酒酵母（芽殖酵母）的 20S、19S、26S 和 30S 蛋白酶体的以下质量： m (20S) = 738.8 ± 2.9 kDa， m (19S) = 926.2 ± 4.8 kDa， m ( 26S) = 1,637.0 ± 7.6 kDa， m (30S) = 2,534.2 ± 10.8 kDa。在某些条件下，观察到较大的 (20S) _x （其中x = 1 至 ∼13）组装体； 19S 调节颗粒也会寡聚，但程度较小，形成 (19S) _x复合物（其中x = 1 至 4，有利于x = 3 三聚体）。 (20S) _x寡聚物在体外受到青睐，因为溶液的 pH 值降低（在 20 mM 醋酸铵溶液中，从 7.0 降至 5.4），并且可能与在碳饥饿下观察到的体内蛋白酶体储存颗粒有关。从m (20S) _x ( x = 1 至 ∼13) 个物种的测量来看，每个多聚体似乎保留了 20S 复合亚基的所有 28 个蛋白质。考虑了可能解释 (20S) _x组件形成的几种结构类型。我们强调每种结构类型[假设的平面、筏状几何形状（其中单个蛋白酶体通过并排相互作用结合）；细长的棒状几何形状（其中子单元首尾相连）；和大致球形的几何形状（通过非特异性亚基相互作用聚集而产生）]是高度推测性的，但考虑起来仍然很有趣，并且提供了简短的讨论。讨论了 CDMS 在表征蛋白酶体和相关寡聚物方面的实用性。

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CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH†

Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S)_x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S)_x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S)_x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S)_x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S)_x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound end-to-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.

更新日期：2023-08-08

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