Puangpila Chanida, El Rassi Ziad
Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071, United States.
Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 May 1;989:112-21. doi: 10.1016/j.jchromb.2015.03.006. Epub 2015 Mar 14.
This research reports a proof-of-concept that describes an instrumental approach that is gel free and label free at both the separation and mass spectrometry ends for the capturing and identification of differentially expressed proteins (DEPs) in diseases, e.g., cancers. The research consists of subjecting/processing equalized and non-equalized (i.e., untreated) disease-free and hepatocellular carcinoma (HCC) human sera via a multicolumn platform for capturing/fractionating human serum fucome. The equalization was performed via the combinatorial peptide ligand library (CPLL) beads technology that ensured narrowing the protein concentration range, thus allowing the detection of low abundance proteins. The equalized and non-equalized disease-free and HCC sera were first fractionated online onto two lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) followed by the online fractionation of the lectin captured fucome by reversed phase chromatography. The online desalted fractions were first subjected to trypsinolysis and then to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. In comparison with untreated serum, the CPLL treated serum is superior in terms of the total number of identified DEPs, which reflected an increased number of DEPs in a wide abundance range. The DEPs in HCC serum were found to be 70 and 40 in both LTA and AAL fractions for the serum treated by CPLL and untreated serum, respectively. In addition, the platform combined with the CPLL treatment was accomplished with virtually no sample loss and dilution as well as with no experimental biases and sample labeling when comparing the diseased-free and cancer sera using LC-MS/MS.
本研究报告了一种概念验证，其描述了一种仪器方法，该方法在分离和质谱两端均无凝胶且无标记，用于捕获和鉴定疾病（如癌症）中差异表达的蛋白质（DEP）。该研究包括通过多柱平台对均衡和不均衡（即未处理）的无病和肝细胞癌（HCC）人血清进行处理，以捕获/分离人血清岩藻糖组。均衡是通过组合肽配体库（CPLL）磁珠技术进行的，该技术确保缩小蛋白质浓度范围，从而能够检测低丰度蛋白质。首先将均衡和不均衡的无病和HCC血清在线分离到两种对岩藻糖特异的凝集素柱上，即橙黄银耳凝集素（AAL）和四角豆凝集素（LTA），然后通过反相色谱对凝集素捕获的岩藻糖组进行在线分离。在线脱盐后的馏分首先进行胰蛋白酶消化，然后进行液相色谱-质谱联用（LC-MS/MS）分析。与未处理的血清相比，CPLL处理的血清在鉴定出的DEP总数方面更具优势，这反映了在广泛丰度范围内DEP数量的增加。对于CPLL处理的血清和未处理的血清，在LTA和AAL馏分中，HCC血清中的DEP分别为70个和40个。此外，当使用LC-MS/MS比较无病血清和癌症血清时，结合CPLL处理的平台几乎没有样品损失和稀释，也没有实验偏差和样品标记。
