Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards.
Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards.
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DOI：
10.1371/journal.pone.0140097
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Aerts JM
中科院分区：
文献类型：
--
作者：
Kramer G;Woolerton Y;van Straalen JP;Vissers JP;Dekker N;Langridge JI;Beynon RJ;Speijer D;Sturk A;Aerts JM
使用质谱进行定量蛋白质组分析对于同时定量各种生物样品（例如人血浆）中的蛋白质具有广阔的前景。迄今为止，针对人血浆中内源蛋白质浓度的可重复测量的研究主要集中于使用同位素标记标准品的靶向分析。另一方面，非靶向蛋白质组学在这方面的应用较少，尽管它在发现蛋白质组学方面发挥了重要作用，在多个研究领域生成了大型数据集。使用非靶向质谱分析 (LCMSE)，我们对来自 30 名健康志愿者的人血浆样本和一份血清样本 (ProteomeXchange：PXD000347) 中丰富的血浆蛋白（43 mg/mL-40 ug/mL 范围）进行了定量。通过使用单一内标估计蛋白质浓度的无标记质谱法获得定量结果。该方法得出了 59 种蛋白质的定量结果（截止 ≥11 个定量样品），其中 41 种蛋白质在所有 31 个样品中进行了定量，其中 23 个样品的测定间变异性≤ 20%。将 7 种载脂蛋白的结果与使用同位素标记标准品获得的结果进行比较，同时将 12 种蛋白质的结果与常规免疫测定进行比较。通过 LCMSE 和免疫测定获得的定量数据的比较显示，12 种测试蛋白质中的 8 种在相对蛋白质丰度 (r = 0.72–0.96) 和可比中值浓度方面具有良好至极好的相关性。与文献中的参考浓度相比，LCMSE 测定的 56 种蛋白质的血浆浓度与靶向研究报告的血浆浓度和同位素标记标准品定量的 7 种载脂蛋白的准确性相似。这项研究表明，LCMSE 可以很好地定量相对丰度，并合理估计丰富血浆蛋白的浓度。
Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research. Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL—40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72–0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature. This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins.