The effects of MS1 injection time, MS2 injection time, dynamic exclusion time, intensity threshold, and isolation width were investigated on the numbers of peptide and protein identifications for single-shot bottom-up proteomics analysis using CZE-MS/MS analysis of a Xenopus laevis tryptic digest. An electrokinetically pumped nanospray interface was used to couple a linear-polyacrylamide coated capillary to a Q Exactive HF mass spectrometer. A sensitive method that used a 1.4 Th isolation width, 60,000 MS2 resolution, 110 ms MS2 injection time, and a top 7 fragmentation produced the largest number of identifications when the CZE loading amount was less than 100 ng. A programmable autogain control method (pAGC) that used a 1.4 Th isolation width, 15,000 MS2 resolution, 110 ms MS2 injection time, and top 10 fragmentation produced the largest number of identifications for CZE loading amounts greater than 100 ng; 7218 unique peptides and 1653 protein groups were identified from 200 ng by using the pAGC method. The effect of mass spectrometer conditions on the performance of UPLC-MS/MS was also investigated. A fast method that used a 1.4 Th isolation width, 30,000 MS2 resolution, 45 ms MS2 injection time, and top 12 fragmentation produced the largest number of identifications for 200 ng UPLC loading amount (6025 unique peptides and 1501 protein groups). This is the first report where the identification number for CZE surpasses that of the UPLC at the 200 ng loading level. However, more peptides (11476) and protein groups (2378) were identified by using UPLC-MS/MS when the sample loading amount was increased to 2 μg with the fast method. To exploit the fast scan speed of the Q-Exactive HF mass spectrometer, higher sample loading amounts are required for single-shot bottom-up proteomics analysis using CZE-MS/MS.

中文翻译：

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质谱参数优化提高毛细管区带电泳单次自底向上蛋白质组学分析的鉴定性能

使用 CZE-MS/MS 对非洲爪蟾进行单次自底向上蛋白质组学分析，研究了 MS1 注射时间、MS2 注射时间、动态排除时间、强度阈值和分离宽度对肽和蛋白质鉴定数量的影响laevis 胰蛋白酶消化。使用电动泵送纳米喷雾接口将线性聚丙烯酰胺涂层毛细管连接到 Q Exactive HF 质谱仪。当 CZE 上样量小于 100 ng 时，使用 1.4 Th 隔离宽度、60,000 MS2 分辨率、110 ms MS2 进样时间和前 7 种碎裂的灵敏方法产生了最多的鉴定结果。使用 1.4 Th 隔离宽度、15,000 MS2 分辨率、110 ms MS2 进样时间的可编程自动增益控制方法 (pAGC)，前 10 个碎片对大于 100 ng 的 CZE 加载量产生了最多的识别；使用 pAGC 方法从 200 ng 中鉴定出 7218 个独特的肽段和 1653 个蛋白质组。还研究了质谱仪条件对 UPLC-MS/MS 性能的影响。使用 1.4 Th 分离宽度、30,000 MS2 分辨率、45 ms MS2 进样时间和前 12 条碎裂的快速方法对 200 ng UPLC 上样量（6025 个独特肽段和 1501 个蛋白质组）产生了最多的鉴定结果。这是 CZE 识别号在 200 ng 负载水平上超过 UPLC 识别号的第一份报告。然而，当使用快速方法将上样量增加到 2 μg 时，使用 UPLC-MS/MS 鉴定了更多的肽 (11476) 和蛋白质组 (2378)。