Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers
Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers
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DOI：
10.1002/pmic.201900192
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发表时间：
2019-12-01
期刊：
影响因子：
3.4
通讯作者：
Voelker, Uwe
中科院分区：
文献类型：
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作者：
Blankenburg, Sascha;Hentschker, Christian;Voelker, Uwe
蛋白质组学分析经常受到可用起始材料数量少的阻碍，例如从体内环境中获得的细菌细胞数量少。在这里，单锅固相强化样品制备（SP3）方案被改编并结合有效的细胞破坏，使用洗涤剂进行细菌蛋白质组分析，数量有限。使用该优化方案，可以显著提高不同革兰氏阳性和革兰氏阴性菌种的肽和蛋白质鉴定，并确保可靠的定量。这种改进的方法与已经建立的金黄色葡萄球菌、猪链球菌和嗜肺军团菌的菌株特异性样品处理方案进行了比较。对嗜肺乳杆菌的蛋白质和多肽的鉴定分别提高了300%和620%，达到了物种特异性鉴定的最高水平。这种增加伴随着蛋白质定量的可重复性和重复之间数据完整性的改善。因此，该方案对从感染分析到环境样品的不同设置的低细菌细胞数进行全面的蛋白质组学分析很有兴趣。
Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid-phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram-positive and Gram-negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain-specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species-specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.