Over 4100 protein identifications from a Xenopus laevis fertilized egg digest using reversed-phase chromatographic prefractionation followed by capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry analysis
Over 4100 protein identifications from a Xenopus laevis fertilized egg digest using reversed-phase chromatographic prefractionation followed by capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry analysis
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DOI：
10.1002/pmic.201600262
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发表时间：
2016-12-01
期刊：
影响因子：
3.4
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Yan, Xiaojing;Sun, Liangliang;Dovichi, Norman J.
从非洲爪哇受精胚胎中产生的胰酶消化液用RPLC进行分离。其中一组30个组分用100min的CZE-ESI-MS/MS分离(总仪器时间50h)，第二组15个组分用3h的UPLC-ESI-MS/MS分离(总仪器时间45h)。CZE-MS/MS产生的蛋白质ID(4134比5787)和多肽ID(22比36 848)是UPLC-MS/MS的70%(比5787)和60%(比UPLC-MS/MS相似的仪器时间(50h比45h)，但总进样量少50倍(1.5mg比75mg)。令人惊讶的是，CZE对两种技术鉴定的多肽产生的峰强度比UPLC高25%，尽管负载量低50倍；这种高灵敏度反映了CZE中使用的电动泵浦纳米螺旋界面产生的高效电离。本文首次比较了CZE-MS/MS和UPLC-MS/MS在大规模真核蛋白质组分析中的应用。CZE-ESI-MS/MS产生的蛋白质和多肽鉴定数目接近UPLC-MS/MS产生的数目，但样本量减少了近两个数量级。
A tryptic digest generated from Xenopus laevis fertilized embryos was fractionated by RPLC. One set of 30 fractions was analyzed by 100-min CZE-ESI-MS/MS separations (50 h total instrument time), and a second set of 15 fractions was analyzed by 3-h UPLC-ESI-MS/MS separations (45 h total instrument time). CZE-MS/MS produced 70% as many protein IDs (4134 versus 5787) and 60% as many peptide IDs (22 535 versus 36 848) as UPLC-MS/MS with similar instrument time (50 h versus 45 h) but with 50 times smaller total consumed sample amount (1.5 mu g versus 75 mu g). Surprisingly, CZE generated peaks that were 25% more intense than UPLC for peptides that were identified by both techniques, despite the 50-fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically pumped nanospray interface used in CZE. This report is the first comparison of CZE-MS/MS and UPLC-MS/MS for large-scale eukaryotic proteomic analysis. The numbers of protein and peptide identifications produced by CZE-ESI-MS/MS approach those produced by UPLC-MS/MS, but with nearly two orders of magnitude lower sample amounts.