Efficient separations of intact proteins using slip-flow with nano-liquid chromatography-mass spectrometry.
Efficient separations of intact proteins using slip-flow with nano-liquid chromatography-mass spectrometry.
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DOI：
10.1021/ac403233d
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发表时间：
2014-02-04
影响因子：
7.4
通讯作者：
Wirth, Mary J.
中科院分区：
文献类型：
--
作者：
Wu, Zhen;Wei, Bingchuan;Zhang, Ximo;Wirth, Mary J.
在反相液相色谱质谱(LC-MS)分离完整蛋白质时，毛细管尖端拉动，密实填充0.47μm的二氧化硅颗粒，具有较高的峰容量和灵敏度。对于C18键合相，滑移流使流动增强了10倍，以允许稳定的纳米喷雾，柱长为4厘米。研究了模型蛋白：核糖核酸酶A、胰酶抑制剂和碳酸氢酶，后者含有超氧化物歧化酶和泛素等杂质。蛋白质在室温下分离良好，峰拖尾可以忽略不计。基于对整个峰的高斯拟合，泛素的峰容量对于10分钟的梯度是195，对于40分钟的梯度是315，而不是外推半最大值的全宽度。经60min梯度分离的细胞裂解物对多肽和相对均一的蛋白质的峰容量极高，达到750以上。获得了每个模型蛋白质的清洁、低噪声的质谱图。峰的物理宽度比传统柱子窄一个数量级，从而提高了灵敏度。从峰形和质谱图可以看出，除泛素外的所有蛋白质都表现出明显的异质性，这是由于蛋白质的多种形态所致。色谱图具有良好的保留时间重现性，相对标准偏差为0.09~0.34%。结果表明，在自上而下的蛋白质组学研究中，亚微米颗粒有望改善LC的分离尺寸。
A capillary with a pulled tip, densely packed with silica particles of 0.47 μm in diameter, is shown to provide higher peak capacity and sensitivity in the separation of intact proteins by reversed-phase liquid chromatography–mass spectrometry (LC–MS). For a C18 bonded phase, slip flow gave a 10-fold flow enhancement to allow for stable nanospray with a 4-cm column length. Model proteins were studied: ribonuclease A, trypsin inhibitor, and carbonic anhydrase, where the latter had impurities of superoxide dismutase and ubiquitin. The proteins were well separated at room temperature with negligible peak tailing. The peak capacity for ubiquitin was 195 for a 10-min gradient and 315 for a 40-min gradient based on Gaussian fitting of the entire peak, rather than extrapolating the full-width at half-maximum. Separation of a cell lysate with a 60 min gradient showed extremely high peak capacities of 750 and above for a peptide and relatively homogeneous proteins. Clean, low noise mass spectra for each model protein were obtained. The physical widths of the peaks were an order of magnitude narrower than those of conventional columns, giving increased sensitivity. All proteins except ubiquitin exhibited significant heterogeneity apparently due to multiple proteoforms, as indicated by both peak shapes and mass spectra. The chromatograms exhibited excellent reproducibility in retention time, with relative standard deviations of 0.09 to 0.34%. The results indicate that submicrometer particles are promising for improving the separation dimension of LC in top-down proteomics.
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影响因子：
4.1
作者：
Eeltink, Sebastiaan;Wouters, Bert;Treumann, Achim
通讯作者：
Treumann, Achim
影响因子：
7.4
作者：
Wei, Bingchuan;Malkin, Douglas S.;Wirth, Mary J.
通讯作者：
Wirth, Mary J.
影响因子：
4.1
作者：
Everley, Robert A.;Croley, Timothy R.
通讯作者：
Croley, Timothy R.
影响因子：
4.7
作者：
Silveyra, MX;Cuadrado-Corrales, N;Sáez-Valero, J
通讯作者：
Sáez-Valero, J
影响因子：
64.8
作者：
Tran, John C.;Zamdborg, Leonid;Ahlf, Dorothy R.;Lee, Ji Eun;Catherman, Adam D.;Durbin, Kenneth R.;Tipton, Jeremiah D.;Vellaichamy, Adaikkalam;Kellie, John F.;Li, Mingxi;Wu, Cong;Sweet, Steve M. M.;Early, Bryan P.;Siuti, Nertila;LeDuc, Richard D.;Compton, Philip D.;Thomas, Paul M.;Kelleher, Neil L.
通讯作者：
Kelleher, Neil L.