Optimization of proteomics sample preparation for identification of host and bacterial proteins in mouse feces.
Optimization of proteomics sample preparation for identification of host and bacterial proteins in mouse feces.
复制标题
DOI：
10.1007/s00216-022-03885-z
复制
发表时间：
2022-03
影响因子：
4.3
通讯作者：
Wysocki, Vicki H.
中科院分区：
文献类型：
--
作者：
Baniasad, Maryam;Kim, Yongseok;Shaffer, Michael;Sabag-Daigle, Anice;Leleiwi, Ikaia;Daly, Rebecca A.;Ahmer, Brian M. M.;Wrighton, Kelly C.;Wysocki, Vicki H.
自下而上的蛋白质组学是研究小鼠肠道菌群功能的有力方法。迄今为止，大多数自下而上的小鼠肠道蛋白质组学研究依赖于有限数量的粪便样本。对于质量有限的样品，这种分析的性能高度依赖于蛋白质提取方案和污染物去除策略。在这里，系统地评价了蛋白质提取方案（使用不同的裂解缓冲液）和污染物去除策略（使用不同类型的过滤器和珠），以最大限度地提高定量再现性和鉴定的蛋白质的数量。总体而言，我们的研究结果建议使用十二烷基硫酸钠（SDS）和尿素在Tris-HCl中的组合的蛋白质提取方法，以产生最大数量的蛋白质鉴定。这些条件导致从革兰氏阳性细菌如厚壁菌门和放线菌中鉴定的蛋白质数量增加，这是一项具有挑战性的任务。我们的分析进一步证实了这些条件导致非丰富的细菌门如变形菌门的提取。此外，我们发现，当与我们优化的提取方法结合时，悬浮液捕获器（S-Trap）的性能优于其他污染物去除方法，提供了可重复性最高的方法，同时产生了最多数量的蛋白质鉴定。总的来说，我们优化的样品制备工作流程简单快速，只需最少的样品处理。此外，我们的方法不需要大量的粪便样本，这是蛋白质组学研究中的一个重要考虑因素，其中小鼠由于特定的生理条件而产生少量的粪便。我们的最终方法提供了对小鼠粪便材料的有效消化，是可重复的，并导致宿主和微生物组蛋白的高蛋白质组覆盖率。
Bottom-up proteomics is a powerful method for the functional characterization of mouse gut microbiota. To date, most of the bottom-up proteomics studies of the mouse gut rely on limited amounts of fecal samples. With mass-limited samples, the performance of such analyses is highly dependent on the protein extraction protocols and contaminant removal strategies. Here, protein extraction protocols (using different lysis buffers) and contaminant removal strategies (using different types of filters and beads) were systematically evaluated to maximize quantitative reproducibility and the number of identified proteins. Overall, our results recommend a protein extraction method using a combination of sodium dodecyl sulfate (SDS) and urea in Tris–HCl to yield the greatest number of protein identifications. These conditions led to an increase in the number of proteins identified from gram-positive bacteria, such as Firmicutes and Actinobacteria, which is a challenging task. Our analysis further confirmed these conditions led to the extraction of non-abundant bacterial phyla such as Proteobacteria. In addition, we found that, when coupled to our optimized extraction method, suspension trap (S-Trap) outperforms other contaminant removal methods by providing the most reproducible method while producing the greatest number of protein identifications. Overall, our optimized sample preparation workflow is straightforward and fast, and requires minimal sample handling. Furthermore, our approach does not require high amounts of fecal samples, a vital consideration in proteomics studies where mice produce smaller amounts of feces due to a particular physiological condition. Our final method provides efficient digestion of mouse fecal material, is reproducible, and leads to high proteomic coverage for both host and microbiome proteins.
登录
查看更多内容
影响因子：
8.8
作者：
Lichtman JS;Ferreyra JA;Ng KM;Smits SA;Sonnenburg JL;Elias JE
通讯作者：
Elias JE
影响因子：
14.9
作者：
Deutsch EW;Csordas A;Sun Z;Jarnuczak A;Perez-Riverol Y;Ternent T;Campbell DS;Bernal-Llinares M;Okuda S;Kawano S;Moritz RL;Carver JJ;Wang M;Ishihama Y;Bandeira N;Hermjakob H;Vizcaíno JA
通讯作者：
Vizcaíno JA
DOI：
10.1146/annurev-anchem-071015-041535
发表时间：
2016-01-01
期刊：
ANNUAL REVIEW OF ANALYTICAL CHEMISTRY, VOL 9
影响因子：
--
作者：
Gillet, Ludovic C.;Leitner, Alexander;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi
影响因子：
3.7
作者：
Kaiser, Brooke L. Deatherage;Li, Jie;Adkins, Joshua N.
通讯作者：
Adkins, Joshua N.
影响因子：
14.8
作者：
Isaacson, Tal;Damasceno, Cynthia M. B.;Rose, Jocelyn K. C.
通讯作者：
Rose, Jocelyn K. C.