白蛋白和免疫球蛋白移除試劑對血清蛋白質體學分析的影響
Abstract
血清為常見尋找生物標記物的來源，其中含有許多高含量的蛋白質，例如：白蛋白和免疫球蛋白。但由於高含量蛋白質於質譜分析中會遮蔽低含量蛋白質的訊號，我們通常會將其移除以利之後的分析。許多市售的親和性管柱可以有效的移除這些高含量的蛋白質，但使用的同時，部分和管柱沒有專一性的蛋白質也可能一併被移除，最終影響血清中蛋白質定量和定性的結果。本次實驗目的為使用Sigma 販售的ProteoPrep® 免疫親和性白蛋白和免疫球蛋白移除管柱，收集其濾液和沖提液搭配質譜分析技術，檢視經過移除高含量蛋白質的步驟對血清蛋白質體學分析的影響。我們將沖提液載入裝有Equalizer固定相的管柱，藉此降低高含量蛋白質與低含量蛋白質之間量的差異。另一分面，我們將濾液使用於等電點聚焦分離儀，藉此降低樣品的複雜度，並比較等電點聚焦分離方法運用於蛋白質層次與胜肽層次之解析度和所鑑定到的蛋白質個數。結果顯示，雖然兩者的解析度差異不大，但利用胜肽等電點聚焦分離方法可以多鑑定到54個蛋白質。血清蛋白質體學分析中，194個蛋白質鑑定於沖提液中而239個蛋白質鑑定於濾液中，最終有116個蛋白質同時被鑑定於兩者。為了確定經過免疫親和性管柱是否影響定量結果，我們挑選Complement C和 Alpha-2-Macroglobulin為例子，利用質譜多反應監測法進行定量分析。結果顯示，Complement C3在沖提液中的含量約占此蛋白質於血清中總量的百分之四左右，而Alpha-2-Macroglobulin在沖提液中的含量約占此蛋白質於血清中總量的百分之十左右。因此藉由本次實驗，我們發現經由移除高含量蛋白質的步驟，的確會影響血清中蛋白質的定性和定量的結果。
Since albumin and IgG are two of the most abundant proteins in human serum, it is being removed in many MS-based assays for the detection of other low abundance proteins. By using commercially available kits, high abundance proteins in serum can be depleted efficiently. However, while removing these abundant proteins, it will also result in losing co-depleted proteins and possibly affecting the overall profile in quantitative proteomics. In this study, ProteoPrep® Immunoaffinity Albumin and IgG Depletion column (Sigma) was used; both elution and flow through fractions were preserved. Equalizer technology was applied on the elution fraction by removing the excess amount of high abundance proteins and contributed to concentrating the low abundance proteins. Besides, flow through fraction was divided into two portions. One was performed protein-level sIEF fractionation and the other was carried out peptide-level sIEF fractionation. Although the resolutions of two sIEF experiments were comparable, a total of 54 more proteins were identified in peptide-level sIEF fractionation. Serum proteome characterization with depletion kit treatment was achieved by making the comparison of protein identification between the elution fraction and the flow through fraction. One hundred and ninety four co-depleted proteins were identified and 116 of them were also detected in the flow through fraction. For example, Complement C3 and Alpha-2-Macroglobulin were two of the co-depleted proteins. By using MRM quantification approach, Complement C3 in the elution fraction was 4.3 % of its overall quantity and Alpha-2-Macroglobulin in the elution fraction was 10.5 % of its overall quantity. The result shows that with the immunodepletion procedure, proteins were nonspecifically co-depleted, leading to effects on protein quantitation and identification consequently.
Since albumin and IgG are two of the most abundant proteins in human serum, it is being removed in many MS-based assays for the detection of other low abundance proteins. By using commercially available kits, high abundance proteins in serum can be depleted efficiently. However, while removing these abundant proteins, it will also result in losing co-depleted proteins and possibly affecting the overall profile in quantitative proteomics. In this study, ProteoPrep® Immunoaffinity Albumin and IgG Depletion column (Sigma) was used; both elution and flow through fractions were preserved. Equalizer technology was applied on the elution fraction by removing the excess amount of high abundance proteins and contributed to concentrating the low abundance proteins. Besides, flow through fraction was divided into two portions. One was performed protein-level sIEF fractionation and the other was carried out peptide-level sIEF fractionation. Although the resolutions of two sIEF experiments were comparable, a total of 54 more proteins were identified in peptide-level sIEF fractionation. Serum proteome characterization with depletion kit treatment was achieved by making the comparison of protein identification between the elution fraction and the flow through fraction. One hundred and ninety four co-depleted proteins were identified and 116 of them were also detected in the flow through fraction. For example, Complement C3 and Alpha-2-Macroglobulin were two of the co-depleted proteins. By using MRM quantification approach, Complement C3 in the elution fraction was 4.3 % of its overall quantity and Alpha-2-Macroglobulin in the elution fraction was 10.5 % of its overall quantity. The result shows that with the immunodepletion procedure, proteins were nonspecifically co-depleted, leading to effects on protein quantitation and identification consequently.
Description
Keywords
白蛋白和免疫球蛋白移除, 等電點聚焦分離方法, equalizer 固定相, 共同移除蛋白, 質譜, albumin and IgG depletion, solution-IEF, equalizer technology, co-depleted proteins, mass spectrometry