疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。

Fast separation and preparation of proteomics samples of human serum using high performance hydrophobic interaction chromatographic cake

A method for the fast separation of human serum and enrichment of low abundance proteins was developed using offline 2D liquid chromatography (2D-LC) consisted of chromatographic cake (10 mm x 20 mm i. d.) and reversed-phase liquid chromatography (RPLC). The protein after separation and enrichment was detected using matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS). This method was validated by four standard proteins at very low concentration. It was found that the detection limits were 1 pmol/microL for the enriched cytochrome-c and myoglobin, and 0.1 pmol/microL for enriched lysozyme and insulin. This method has been applied to the proteomic research of human serum, and it was found that the signal intensity and the number of detected proteins/peptides in MALDI-TOF MS increased with the increase of the loading sample volume of the human serum on chromatographic cake. A total of 285 fractions (M(r) < 15 000) were found when 1.0 mL serum sample was loaded on the chromatographic cake. In addition, cytochrome-c in low abundance was also separated and enriched successfully when 1 microg cytochrome-c was added into 0.5 mL original serum. The results showed that 2D-LC consisting of the chromatographic cake and RPLC was successfully applied to not only the fast separation and preparation of human serum sample with large loading volume in one cycle of analysis, but also the efficient isolation and enrichment of the lower abundance proteins/peptides in human serum. Moreover, it successfully increased the detection efficiency of the low abundance proteins/peptides in human serum with MALDI-TOF MS.