Investigation of an optimal cell lysis method for the study of the zinc metalloproteome of Histoplasma capsulatum.
Investigation of an optimal cell lysis method for the study of the zinc metalloproteome of Histoplasma capsulatum.
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DOI：
10.1007/s00216-017-0556-7
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发表时间：
2017-10
影响因子：
4.3
通讯作者：
Vonderheide AP
中科院分区：
文献类型：
--
作者：
Donnell AM;Lewis S;Abraham S;Subramanian K;Figueroa JL;Deepe GS Jr;Vonderheide AP
本工作旨在评估最佳提取条件的研究中的金属蛋白质组的二型真菌荚膜组织胞浆菌。人体对H.荚膜梭菌感染是宿主巨噬细胞（MMCs）内锌的隔离，如Vignesh等人（Immunity 39：697-710，2013）和Vignesh等人（PLOS Pathog 9：E1003815，2013）所报道。因此，含锌的金属蛋白是最感兴趣的，因为它在真菌的生存中起着关键作用。金属蛋白质组学中的一个挑战是保留蛋白质的天然结构以保留非共价结合的金属。蛋白质组学中的许多常规细胞裂解、分离和鉴定技术是在可能导致蛋白质变性的条件下进行的。研究了各种细胞裂解技术，以努力在裂解和随后的分析期间保持金属蛋白，同时，使其足够强以打破细胞壁，从而允许进入胞质金属蛋白。还研究了向裂解缓冲液中加入1%Triton x-100（一种非离子去污剂）。考虑了7种裂解方法，包括：玻璃匀浆器（H）、珠搅拌器（BB）、超声处理探针（SP）、含1% Triton x-100的涡旋（V，T）、无Triton x-100的涡旋（V，NT）、超声处理浴、涡旋和1% Triton x-100（SB，V，T）以及超声处理浴、涡旋和无Triton x-100（SB，V，NT）。使用Qubit®测定来比较总蛋白质浓度，并使用电感耦合等离子体质谱法（ICP-MS）来分析细胞裂解物的总金属。尺寸排阻色谱耦合电感耦合等离子体质谱（SEC-HPLC-ICP-MS）用于分离的金属蛋白在细胞裂解物和锌的浓度在一个宽的分子量范围内进行了检查。还考虑了其他因素，如潜在的污染源。涉及涡旋H.在1%Tritonx-100裂解缓冲液中用500 μm玻璃珠（V，T）对荚膜酵母细胞进行离心，发现最有利于提取完整的锌金属蛋白，其表现为最高的锌蛋白比（1.030 ng Zn/μg蛋白），以及Zn在高、中、低分子量之间的分布，表明蛋白质变性量最小。
This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body’s responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697–710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma–mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 μm glass beads in a 1% Triton x-100 lysis buffer (V, T) was found to be most advantageous to extract intact zinc metalloproteins as demonstrated by the highest Zn to protein ratio, 1.030 ng Zn/μg protein, and Zn distribution among high, mid, and low molecular weights suggesting the least amount of protein denaturation.
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影响因子：
3.2
作者：
Garcera, Ana;Casas, Celia;Herrero, Enrique
通讯作者：
Herrero, Enrique
影响因子：
2.7
作者：
Liu, Chang;Apodaca, Jennifer;Rao, Hai
通讯作者：
Rao, Hai
影响因子：
3.5
作者：
BLECHL, AE;THRASHER, KS;GREENE, FC
通讯作者：
GREENE, FC
影响因子：
6.1
作者：
de Magalhaes, Cristiana Schmidt;Zezzi Arruda, Marco Aurelio
通讯作者：
Zezzi Arruda, Marco Aurelio
影响因子：
2.6
作者：
HORVATH, A;RIEZMAN, H
通讯作者：
RIEZMAN, H