Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver V5Z 4H4, Canada.
Michael Cuccione Childhood Cancer Research Program, BC Children's Hospital, Vancouver V5Z 4H4, Canada.
J Proteome Res. 2021 May 7;20(5):3002-3008. doi: 10.1021/acs.jproteome.0c01049. Epub 2021 Mar 29.
The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin-labeled peptide. Recent advances in labeling have led to an increase in biotinylation efficiency and shifted the interest to the detection of the site of biotinylation. This has increased the confidence in identification and provided additional structural information, yet it requires the efficient release of the biotinylated protein/peptide and the sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics, the effect of biotinylation on the chromatographic, ionization, and fragmentation behavior and the ultimate detection of peptides is not well understood. To address this, we compare two commercially available biotin labels, EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase in the hydrophobicity and charge reduction with an increasing number of biotin labels attached. On the basis of our data, we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.
生物素与链霉亲和素的高亲和力使其成为蛋白质组学中应用最广泛的亲和标签之一。早期的方法仅使用生物素来进行富集，并且大多忽略了带有生物素标记的肽。最近在标记方面的进展提高了生物素化效率，并将研究重点转移到生物素化位点的检测上。这提高了鉴定的可信度，并提供了额外的结构信息，但需要有效地释放生物素化的蛋白质/肽，并通过 LC-MS/MS 对生物素化肽进行灵敏的分离和检测。尽管生物素在亲和蛋白质组学中的应用已有很长时间，但生物素化对肽的色谱、离子化和片段化行为以及最终检测的影响尚未得到很好的理解。为了解决这个问题，我们比较了两种市售的生物素标签，EZ-Link Sulfo-NHS-Biotin 和 Sulfo-NHS-SS-Biotin，后者含有不稳定的连接子，可以有效地将生物素释放出来，以确定肽修饰对肽检测的影响。我们发现，随着连接的生物素标签数量的增加，肽的疏水性增加，电荷减少。基于我们的数据，我们建议进行梯度优化，以考虑到更疏水的生物素化肽，并包括单价前体，以解释生物素引起的电荷减少。
