Filter-aided sample preparation (FASP) remains a popular choice for proteomic sample preparation, particularly for its ability to produce a ‘clean’ peptide sample clear of large molecule contaminants. However, sample loss continues to be a problem particularly for sample inputs that contain less than ten micrograms of protein. Here, we describe that the simple addition of a polymer, polyvinylpyrrolidone-40 (PVP-40) to the protein sample prior to FASP digest significantly improves peptide recovery and identifications, especially with lower level sample inputs. PVP-FASP produces clean samples which required no additional sample clean-up prior to nanoLC-MS analysis. In addition, PVP-FASP is compatible with other FASP modifications, including the use of sodium deoxycholate (DOC) to improve trypsin digestion.

Significance

Simple modification to FASP procedure improves sample recovery during proteomic digests in SDS, improving peptide identifications and median peptide intensity.

中文翻译：

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将聚乙烯吡咯烷酮添加到低水平的蛋白质样品中可显着改善FASP消化液中的肽回收率：对FASP方案的廉价且简单的修改

滤膜辅助样品制备（FASP）仍然是蛋白质组学样品制备的普遍选择，特别是因为它能够产生不含大分子污染物的“干净”肽样品。但是，样品丢失仍然是一个问题，特别是对于包含少于十微克蛋白质的样品输入而言。在这里，我们描述了在FASP消化之前向蛋白质样品中简单添加聚合物聚乙烯吡咯烷酮40（PVP-40）可以显着改善肽的回收率和鉴定效果，尤其是在较低水平的样品输入下。PVP-FASP产生干净的样品，在nanoLC-MS分析之前不需要额外的样品清洁。此外，PVP-FASP与其他FASP修改兼容，包括使用脱氧胆酸钠（DOC）改善胰蛋白酶消化。

意义

对FASP程序的简单修改可改善SDS中蛋白质组学消化过程中的样品回收率，改善肽段鉴定和中值肽段强度。