High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics
High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics
复制标题
DOI：
10.1021/ac0202280
复制
发表时间：
2002-08-15
影响因子：
7.4
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Shen, YF;Zhao, R;Smith, RD
我们描述了高效（峰值容量类似于 10(3)）纳米级（使用小至 15 mm 的色谱柱内径）液相色谱 (nanoLC)/低流速电喷雾 (nanoESI) 质谱 (MS)，用于复杂的整体细胞蛋白酶消化（即蛋白质组学）的灵敏分析。使用液体浆料填充方法和精心选择的填充溶剂，在 18 000 psi 的压力下，成功地将 3 微米 C18 键合多孔（300 埃孔）二氧化硅颗粒填充在内径为 14.9-74..5 pm 的 87 厘米长毛细管中。在流动相输送压力为 10 000 psi 的情况下，这些填充毛细管在 0.2 cm/s 的 LC 线速度下提供低至 20 nL/min 的流动相流速，这对于分离效率而言接近最佳。为了保持色谱效率，专门生产了内部通道直径小至 10 pm 的接头，用于将填充毛细管连接到可更换的 nanoESI 发射器 孔径为2-10μm（取决于填充毛细管尺寸）。通过 nanoESI 接口与混合四极杆飞行时间 MS 在线耦合，当使用正反馈切换阀定量引入样品时，nanoLC 分离可为蛋白质组蛋白水解多肽混合物提供类似于 10(3) 的峰值容量。在相对较大的样品负载范围内（例如，内径为 14.9 和 29.7 的毛细管分别为 5-100 ng 和 50-500 ng 细胞蛋白水解肽），发现复杂混合物中低丰度组分的 nanoLC/nanoESI MS 响应随样品负载量线性增加。在 20-400 nL/min 的流量范围内，nanoLC/nanoESI-MS 灵敏度也随着流速的降低而线性增加（或近似与毛细管内径的平方成反比）。因此，除了较低的负载量外，减小分离毛细管内径的效果相当于增加样品负载量，这对于样品有限的蛋白质组学应用非常重要。与无孔颗粒相比，使用表面孔为300埃的多孔C 18 颗粒没有观察到对洗脱多肽的回收率有显着影响。还使用高效 nanoLC 演示了串联 MS 分析。分离。检查了单柱（使用单个 nanoESI 发射器）、不同柱（使用不同 nanoESI 发射器的相同和不同内径）运行之间以及不同样品的色谱、洗脱时间、MS 响应强度和质量测量精度。 （不同浓度的细胞蛋白水解肽 I），并对复杂的蛋白质组样品进行了稳健且可重复的灵敏分析。
We describe high-efficiency (peak capacities of similar to10(3)) nanoscale (using column inner diameters down to 15 mum) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74..5 pm were successfully packed with 3-mum C18-bonded porous (300-Angstrom pores) silica particles at a pressure of 18 000 psi. With a mobile-phase delivery pressure of 10 000 psi, these packed capillaries provided mobile-phase flow rates as low as similar to20 nL/min at LC linear velocities of similar to0.2 cm/s, which is near optimal for separation efficiency., To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 pm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 mum (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of similar to10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-mum-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C 18 particles with surface pores of 300-Angstrom versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC. seprations. Chromatographic, elution time, MS response intensity and Mass. measurement accuracy was examined between runs with a ingle column.(with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples. (various concentrations of cellular proteolytic peptides I) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.