Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-μm-i.d. polystyrene-divinylbenzene monolithic capillary columns
Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-μm-i.d. polystyrene-divinylbenzene monolithic capillary columns
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DOI：
10.1021/ac030163g
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发表时间：
2003-10-15
影响因子：
7.4
通讯作者：
Karger, BL
中科院分区：
文献类型：
--
作者：
Ivanov, AR;Zang, L;Karger, BL
这项工作探讨了 20-mum-i.d 的使用。聚合聚苯乙烯-二乙烯基苯整体纳米毛细管柱，用于胰蛋白酶消化肽混合物的 LC-ESI-MS 分析。与微粒的填充相反，毛细管柱是在熔融石英毛细管中通过苯乙烯和二乙烯基苯的热诱导原位共聚制备的，无需高压。聚合条件和流动相组成针对色谱性能进行了优化，使肽分离效率超过 100 000 板/米。发现使用离子阱 MS 的 MS 和 MS/MS 模式具有高质量灵敏度（类似于 10 amol 的肽），与 75-mum-i.d. 相比，提高了高达 20 倍。纳米柱。实现了较宽的线性动态范围（类似于 4 个数量级），并且发现等度和梯度洗脱分离具有良好的运行间和柱间重现性。使用模型蛋白和组织提取物作为样品。提出了对组织提取物的蛋白水解消化物进行梯度纳米 LC-MS 分析，其样本量相当于注射的 1000 个细胞。
This work explores the use of 20-mum-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100 000 plates/m for peptide separations. High mass sensitivity (similar to10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-mum-i.d. nanocolumns. A wide linear dynamic range (similar to4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of similar to1000 cells injected, is presented.