Wang Nan, Tang Yanan, Chen Lu, Li Liang
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
J Chromatogr A. 2014 Apr 18;1338:51-7. doi: 10.1016/j.chroma.2014.02.050. Epub 2014 Feb 24.
Mass spectrometric profiling of the proteome of a small number of cells requires not only a sensitive instrument for protein/peptide separation and detection, but also a robust sample preparation protocol to process a very small amount of proteins (<1μg) present in few cells. We have developed and evaluated the performance of a microbore liquid chromatography (LC) UV detection system for quantifying the total amount of peptides in a shotgun proteome analysis workflow that is tailored for the analysis of hundreds of cancer cells. Upon the sample injection into a 1-mm-diameter reversed phase column, a step-gradient was used to first remove salts and other impurities and then elute the peptides quickly without much separation. The UV absorbance of eluted peptides at 214nm was used for peptide quantification with the aid of a calibration curve of a tryptic digest of a mixture of four standard proteins. Two linear calibration regions could be obtained in the peptide amount ranging from 0.03μg to 0.3μg and from 0.6μg to 5μg. The limit of quantification (LOQ) was determined to be 30ng (or 39ng in the linear calibration range). However, the presence of background proteins, mainly keratins, introduced during the sample preparation process was found to be the limiting factor in quantifying a lower amount of peptides from a cell lysate digest. With background absorbance from the digest of contaminant proteins in a solution, the LOQ was found to be 200ng. This nondestructive microbore LC-UV method should be useful in assessing sample quality during the development and applications of an efficient sample preparation method for proteome analysis of a small number of cells. As an example, this method was used for quantifying the peptides generated from breast cancer MCF-7 cell extracts with a limited number of cells: 250, 500 and 1000 cells. Using capillary LC quadrupole time-of-flight mass spectrometry, 81-126, 122-154 and 256-282 proteins could be identified from 250, 500, and 1000 cells, respectively, in duplicate experiments. This method was also applied for the analysis of biological triplicate samples of MCF-7 cells. The average numbers of peptides and proteins detected from the experimental triplicate analyses of biological triplicate samples were 400±71 (9 datasets) and 124±14, respectively, from 250 cells, and 531±44 and 162±16, respectively, from 500 cells.
对少量细胞的蛋白质组进行质谱分析，不仅需要一台灵敏的仪器用于蛋白质/肽段的分离和检测，还需要一套强大的样品制备方案来处理少量细胞中存在的极少量蛋白质（<1μg）。我们开发并评估了一种微径液相色谱（LC）紫外检测系统的性能，该系统用于在一种为分析数百个癌细胞量身定制的鸟枪法蛋白质组分析工作流程中定量肽段的总量。将样品注入内径为1mm的反相柱后，采用梯度洗脱，首先去除盐类和其他杂质，然后快速洗脱肽段，分离效果不佳。在四种标准蛋白质混合物的胰蛋白酶消化产物校准曲线的辅助下，利用洗脱肽段在214nm处的紫外吸光度对肽段进行定量。在肽段量为0.03μg至0.3μg以及0.6μg至5μg范围内可获得两个线性校准区域。定量限（LOQ）确定为30ng（在线性校准范围内为39ng）。然而，发现样品制备过程中引入的背景蛋白质（主要是角蛋白）的存在是定量细胞裂解物消化产物中较低量肽段的限制因素。由于溶液中污染物蛋白质消化产物的背景吸光度，LOQ为200ng。这种非破坏性的微径LC-UV方法在开发和应用用于少量细胞蛋白质组分析的高效样品制备方法过程中评估样品质量时应会很有用。例如，该方法用于定量从有限数量的乳腺癌MCF-7细胞提取物中产生的肽段：250、500和1000个细胞。使用毛细管LC四极杆飞行时间质谱，在重复实验中，分别从250、500和1000个细胞中鉴定出81 - 126、122 - 154和256 - 282种蛋白质。该方法还应用于MCF-7细胞的生物重复样品分析。从250个细胞的生物重复样品的实验重复分析中检测到的肽段和蛋白质的平均数量分别为400±71（9个数据集）和124±14，从500个细胞中分别为531±44和162±16。
