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Quantification and Identification of Microproteinuria Using Ultrafiltration and ATR-FTIR Spectroscopy.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-01-15 , DOI: 10.1021/acs.analchem.9b03081 David Perez-Guaita 1 , Zack Richardson , Philip Heraud , Bayden Wood
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-01-15 , DOI: 10.1021/acs.analchem.9b03081 David Perez-Guaita 1 , Zack Richardson , Philip Heraud , Bayden Wood
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The presence of low amounts of specific proteins in urine can be an indicator of diagnosis and prognosis of several diseases including renal failure and cancer. Hence, there is an urgent need for Point-of-care (PoC) methods, which can quantify microproteinuria levels (30-300 ppm) and identify the major proteins associated with the microproteinuria. In this study, we coupled ultracentrifugation with attenuated total reflectance-Fourier transform infrared (ATR-FTIR) to identify and quantify proteins in urine at low parts per million levels. The process involves the preconcentration of proteins from 500 μL of urine using an ultrafiltration device. After several washings, the isolated proteins are dried onto the ATR crystal forming a thin film. Imaging studies showed that the absorbance of the protein bands was linear with the amount of mass deposited on the crystal. The methodology was first evaluated with artificial urine spiked with 30-300 ppm of albumin. The calibration showed acceptable linearity (R2 = 0.97) and a limit of detection of 6.7 ppm. Linear relationships were also observed from urine of healthy subjects spiked with microproteinuria concentrations of albumin, immunoglobulin, and hemoglobin, giving a prediction error of the spiked concentration of 23 ppm. When multiple proteins were spiked into the real urine, multivariate analysis was able to decompose the data set into the different proteins, but the multicomponent evaluation was challenging for proteins at low levels. Although the introduction of a preprocessing step reduces the PoC capability of the method, it largely increases its performance, showing great potential as a tool for the diagnosis and prognosis of several illnesses affecting urine proteic composition.
中文翻译：
使用超滤和ATR-FTIR光谱对微量蛋白尿进行定量和鉴定。
尿液中少量特定蛋白质的存在可以指示诊断和预后多种疾病，包括肾衰竭和癌症。因此，迫切需要即时护理（PoC）方法，该方法可以量化微量蛋白尿水平（30-300 ppm）并鉴定与微量蛋白尿有关的主要蛋白。在这项研究中，我们将超速离心与衰减的全反射-傅立叶变换红外光谱（ATR-FTIR）结合使用，以低百万分之几的浓度鉴定和定量尿液中的蛋白质。该过程涉及使用超滤设备从500μL尿液中预浓缩蛋白质。几次洗涤后，将分离的蛋白质干燥到ATR晶体上，形成薄膜。成像研究表明，蛋白带的吸收率与沉积在晶体上的质量的数量呈线性关系。首先用掺有30-300 ppm白蛋白的人造尿液评估该方法。校准显示出可接受的线性度（R2 = 0.97），检出限为6.7 ppm。从健康受试者的尿液中也观察到线性关系，尿液中掺入了微蛋白尿素的白蛋白，免疫球蛋白和血红蛋白浓度，得出的掺入浓度预测误差为23 ppm。当将多种蛋白质加标到真实尿液中时，多变量分析能够将数据集分解为不同的蛋白质，但是对于低含量的蛋白质，多组分评估具有挑战性。尽管引入预处理步骤会降低该方法的PoC功能，
更新日期：2020-01-16
中文翻译：
使用超滤和ATR-FTIR光谱对微量蛋白尿进行定量和鉴定。
尿液中少量特定蛋白质的存在可以指示诊断和预后多种疾病，包括肾衰竭和癌症。因此，迫切需要即时护理（PoC）方法，该方法可以量化微量蛋白尿水平（30-300 ppm）并鉴定与微量蛋白尿有关的主要蛋白。在这项研究中，我们将超速离心与衰减的全反射-傅立叶变换红外光谱（ATR-FTIR）结合使用，以低百万分之几的浓度鉴定和定量尿液中的蛋白质。该过程涉及使用超滤设备从500μL尿液中预浓缩蛋白质。几次洗涤后，将分离的蛋白质干燥到ATR晶体上，形成薄膜。成像研究表明，蛋白带的吸收率与沉积在晶体上的质量的数量呈线性关系。首先用掺有30-300 ppm白蛋白的人造尿液评估该方法。校准显示出可接受的线性度（R2 = 0.97），检出限为6.7 ppm。从健康受试者的尿液中也观察到线性关系，尿液中掺入了微蛋白尿素的白蛋白，免疫球蛋白和血红蛋白浓度，得出的掺入浓度预测误差为23 ppm。当将多种蛋白质加标到真实尿液中时，多变量分析能够将数据集分解为不同的蛋白质，但是对于低含量的蛋白质，多组分评估具有挑战性。尽管引入预处理步骤会降低该方法的PoC功能，
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