nanoLC-MS/MS检测未成熟树突状细胞与其外泌体蛋白组分差异的初步研究

  目的  采用一种相对快速、样品需求量少的纳升级液相色谱串联质谱（nanoLC-MS/MS）法初步探究未成熟树突状细胞DC2.4及其来源的外泌体（DC-Exo）蛋白组分差异。  方法  收集DC2.4细胞培养基上清，采用梯度离心法分离DC-Exo，使用蔗糖密度梯度超速离心进一步纯化而获得DC-Exo测定样品。采用Bradford法测定其蛋白总量，使用动态光散射法和透射电镜分别对DC-Exo粒度分布和形态进行考察。采用FASP酶解法制备DC2.4细胞和DC-Exo待测蛋白样品。nanoLC-MS/MS检测待测样品:采用μLPickUp上样模式，上样量仅1 μg，Transport liquid及Micro A相均为0.05%三氟乙酸-2%乙腈（V/V）aq.；nanoLC使用Acclaim? PepMap RSLC分析柱，流动相为（A）0.1%甲酸水溶液（V/V）和（B）0.08%甲酸-80%乙腈（V/V），采用梯度洗脱，流速为0.3 μL/min；MS/MS采用LTQ Obitrap双重质谱，使用APCI nanospray离子源，“一拖十”数据采集模式。得到结果以Uniport Mouse (Fasta文件) 为蛋白数据库，采用SQUEST对DC2.4细胞及DC-Exo蛋白质谱信息进行搜索和匹配，并整理分析。  结果  得到产量较高、粒径为40～200 nm的DC-Exo。用FASP酶解法处理得到的DC2.4细胞和DC-Exo冻干蛋白样品复溶后可直接上样，且上样量仅需1 μg。搜库结果显示，DC2.4细胞含蛋白998种，其中高表达227种，特有蛋白535种；DC-Exo仅含蛋白348种，其中特有高表达18种；两者共有蛋白为306种，共有高表达蛋白7种。  结论  本实验采用的nanoLC-MS/MS法所需进样量少，可相对快速地初步检测DC2.4细胞及其外泌体的蛋白组分差异。

Preliminary Investigation on Difference of Protein Compositions Between DC2.4 Cells and Their Derived Exosomes by nanoLC-MS/MS

  Objective  To preliminarily investigate the differences of protein composition between immature dendritic cells (DC2.4) and their derived exosomes (DC-Exo) using a relatively rapid and sample-saving method based on nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS).  Methods  The supernatant of DC2.4 cells culture medium was collected and gradient centrifugation was applied to primarily extract and isolate DC-Exo; then sucrose density gradient ultracentrifugation was adopted to purify the DC-Exo. Bradford protein assay was used to determine the total protein content of the purified DC-Exo, and dynamic light scattering and transmission electron microscope were conducted to characterize the morphology and size distribution of the DC-Exo. Afterwards, protein samples including DC2.4 cells and DC-Exo were prepared by FASP enzymolysis method. Samples were performed nanoLC-MS/MS assay. The μLPickUp sample loading mode was used and only 1 μg of protein sample was required for each assay. The phase of Transport liquid and Micro A were both 0.05% trifluoroacetic acid-2% acetonitrile (ACN) aq. (V/V). Acclaim? PepMap RSLC column was used to separate sample compositions and the gradient elute was adopted where the mobile phase consisted of (A) 0.1% formic acid (FA) and (B) 0.08% FA-80% ACN aq. (V/V) with flow rate of 0.3 μL/min. Positive APCI nanospray interface was used and “one-drive-ten” schema was set to collect primary information. The collected data was then searched and matched based on Uniport Mouse Fasta file as protein database in this case, and the re-annotated data was further sorted out and analyzed.  Results  In the current study, relatively high yield of DC-Exo samples with sizes of 40-200 nm were obtained. The lyophilized protein samples prepared by FASP method could be loaded directly after redissolution, and only 1 μg of protein sample is required. The annotated results showed that DC2.4 cells contained 998 kinds of proteins, among which 227 were highly expressed and 535 were unique; while DC-Exo contained only 348 types of proteins, among which 18 were uniquely and highly expressed. There were 306 kinds of consensus proteins in both DC2.4 cells and DC-Exo, among them 7 kinds were highly expressed.  Conclusion  The nanoLC-MS/MS method developed in this study only requires very small amount of protein samples, and it could primarily differentiate the protein compositions between DC2.4 cells and their derived exosomes rapidly.