Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, United States.
Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, United States.
J Am Soc Mass Spectrom. 2023 Oct 4;34(10):2247-2258. doi: 10.1021/jasms.3c00191. Epub 2023 Sep 20.
Ion activation methods carried out at gas pressures compatible with ion mobility separations are not yet widely established. This limits the analytical utility of emerging tandem-ion mobility spectrometers that conduct multiple ion mobility separations in series. The present work investigates the applicability of collision-induced dissociation (CID) at 1 to 3 mbar in a tandem-trapped ion mobility spectrometer (tandem-TIMS) to study the architecture of protein complexes. We show that CID of the homotetrameric protein complexes streptavidin (53 kDa), neutravidin (60 kDa), and concanavalin A (110 kDa) provides access to all subunits of the investigated protein complexes, including structurally informative dimers. We report on an "atypical" dissociation pathway, which for concanavalin A proceeds via symmetric partitioning of the precursor charges and produces dimers with the same charge states that were previously reported from surface induced dissociation. Our data suggest a correlation between the formation of subunits by CID in tandem-TIMS/MS, their binding strengths in the native tetramer structures, and the applied activation voltage. Ion mobility spectra of in situ-generated subunits reveal a marked structural heterogeneity inconsistent with annealing into their most stable gas phase structures. Structural transitions are observed for in situ-generated subunits that resemble the transitions reported from collision-induced unfolding of natively folded proteins. These observations indicate that some aspects of the native precursor structure is preserved in the subunits generated from disassembly of the precursor complex. We rationalize our observations by an approximately 100-fold shorter activation time scale in comparison to traditional CID in a collision cell. Finally, the approach discussed here to conduct CID at elevated pressures appears generally applicable also for other types of tandem-ion mobility spectrometers.
在与离子淌度分离兼容的气压下进行的离子激活方法尚未得到广泛应用。这限制了新兴串联离子淌度谱仪的分析应用，这些谱仪可以串联进行多次离子淌度分离。本工作研究了在串联俘获离子淌度谱仪（tandem-TIMS）中在 1 至 3 mbar 下进行碰撞诱导解离（CID）的适用性，以研究蛋白质复合物的结构。我们表明，同源四聚体蛋白复合物链霉亲和素（53 kDa）、中性亲和素（60 kDa）和伴刀豆球蛋白 A（110 kDa）的 CID 可获得所研究的蛋白复合物的所有亚基，包括结构信息丰富的二聚体。我们报告了一种“非典型”的解离途径，对于伴刀豆球蛋白 A，通过前体电荷的对称分配进行，产生具有相同电荷状态的二聚体，这是以前从表面诱导解离中报道的。我们的数据表明，在串联-TIMS/MS 中通过 CID 形成亚基与它们在天然四聚体结构中的结合强度以及所应用的激活电压之间存在相关性。原位生成的亚基的离子淌度谱显示出明显的结构异质性，与它们在最稳定的气相结构中的退火不一致。观察到原位生成的亚基发生结构转变，类似于从天然折叠蛋白的碰撞诱导解折叠中报告的转变。这些观察结果表明，在从前体复合物的解组装中产生的亚基中保留了一些天然前体结构的方面。我们通过与传统碰撞池中 CID 相比，激活时间尺度大约缩短了 100 倍，来合理化我们的观察结果。最后，这里讨论的在升高压力下进行 CID 的方法似乎也适用于其他类型的串联离子淌度谱仪。
