Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
J Proteome Res. 2010 Feb 5;9(2):997-1006. doi: 10.1021/pr900888b.
A high-throughput approach and platform using 15 min reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking 20 reference peptides at varying concentrations from 1 ng/mL to 10 microg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected 13 out of the 20 spiked peptides that had concentrations >or=100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for 19 of the 20 peptides with all spiking levels present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects and achieve high measurement accuracy, but in turn limits the achievable dynamic range compared to the IMS-TOF instrument.
采用 15 分钟反相毛细管液相色谱 (RPLC) 分离与离子淌度谱-质谱 (IMS-MS) 联用的高通量方法和平台,用于快速分析复杂蛋白质组学样品。为了测试短 LC 梯度的分离质量,将 20 种参考肽以 1ng/mL 至 10μg/mL 的不同浓度混入小鼠血等离子体的胰蛋白酶消化物中,并用 LC-线性离子阱傅里叶变换 (FT) MS 和 LC-IMS-TOF MS 进行分析。LC-FT MS 检测到 20 个掺入肽中的 13 个,其浓度 > = 100ng/mL。相比之下,LC-IMS-TOF MS 分析中的漂移时间选择质谱可鉴定所有掺入水平存在的 20 个肽中的 19 个。LC-IMS-TOF MS 系统的更大动态范围可归因于两个因素。首先,LC-IMS-TOF MS 系统能够将低浓度掺入肽与高浓度小鼠肽基质成分进行漂移时间分离,从而减少信号干扰和背景,并允许分离出否则会被其他成分掩盖的物质。其次,混合 FT MS 仪器线性离子阱中的自动增益控制 (AGC) 限制了累积的离子数量,以减少空间电荷效应并实现高测量精度,但与 IMS-TOF 仪器相比,这反过来限制了可实现的动态范围。