Peterson Amelia, Hohmann Laura, Huang Li, Kim Bong, Eng Jimmy K, Martin Daniel B
Department of Chemistry, University of Wisconsin, Madison, USA.
J Proteome Res. 2009 Aug;8(8):4161-8. doi: 10.1021/pr9001417.
Substantial energy and resources have been invested in improving mass spectrometry (MS) instrumentation, upstream sample preparation protocols, and database search strategies to maximize peptide and protein identifications. The role of HPLC sample loading methods in maximizing MS identifications has been largely overlooked, and there exists an immense heterogeneity in the methods employed in the proteomics literature. We sought to optimize loading methods by testing multiple loading conditions (buffer composition, resin, initial gradient) using tryptic digests of an 18 protein mixture and whole yeast lysate. The loading buffer acetonitrile (ACN) concentration greatly affected peptide identifications: up to a 26% increase in peptide identifications was observed by decreasing the ACN concentration from 5 to 2% during sample loading. Hydrophilic peptides were the main contributors to the increase in peptide identifications and, at higher ACN concentrations, were washed from the precolumn during desalting. Sampling of the hydrophilic peptides was enhanced by using a shallow initial ACN gradient. The results were found to be resin-specific and not generalizable. Our investigation demonstrates the often unappreciated importance of optimizing sample loading conditions to reflect the aims of the research and the characteristics of the LC configurations employed.
为了最大限度地提高肽段和蛋白质的鉴定率，人们投入了大量的精力和资源来改进质谱（MS）仪器、上游样品制备方案和数据库搜索策略。高效液相色谱（HPLC）进样方法在最大化质谱鉴定方面的作用在很大程度上被忽视了，并且蛋白质组学文献中使用的方法存在极大的异质性。我们试图通过使用18种蛋白质混合物的胰蛋白酶消化物和全酵母裂解物测试多种进样条件（缓冲液组成、树脂、初始梯度）来优化进样方法。进样缓冲液中乙腈（ACN）的浓度对肽段鉴定有很大影响：在进样过程中将ACN浓度从5%降至2%时，肽段鉴定数量最多可增加26%。亲水性肽段是肽段鉴定数量增加的主要贡献者，在较高的ACN浓度下，它们在脱盐过程中会从预柱上被洗脱下来。使用浅的初始ACN梯度可增强亲水性肽段的进样。结果发现这些结果具有树脂特异性，不可推广。我们的研究表明，优化进样条件以反映研究目的和所采用的液相色谱配置的特点，其重要性常常未被重视。
