A MICROSCALE ELECTROSPRAY INTERFACE FOR ONLINE, CAPILLARY LIQUID-CHROMATOGRAPHY TANDEM MASS-SPECTROMETRY OF COMPLEX PEPTIDE MIXTURES
A MICROSCALE ELECTROSPRAY INTERFACE FOR ONLINE, CAPILLARY LIQUID-CHROMATOGRAPHY TANDEM MASS-SPECTROMETRY OF COMPLEX PEPTIDE MIXTURES
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DOI：
10.1021/ac00120a019
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发表时间：
1995-12-15
影响因子：
7.4
通讯作者：
LEE, TD
中科院分区：
文献类型：
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作者：
DAVIS, MT;STAHL, DC;LEE, TD
一个微毛细管液相色谱（HPLC）系统设计用于肽和蛋白质样品的梯度洗脱，现在的速度< 1 μ L/min已耦合到一个三扇区四极质谱仪通过一个简单的无鞘电喷雾接口（微喷雾）。微喷雾接口使用一种未涂覆的熔融石英针，其尖端外径在15-20 μ m的范围内，开口直径小于5 μ m。通过使用集成到针组件中的亲水性PVDF膜过滤器实现在线样品过滤，以防止抽吸针堵塞。喷雾电位（0.5-1 kV）通过毛细管接头直接施加到样品流。在整个梯度范围（0-90%乙腈水溶液）内获得稳定的电喷雾条件，并且通常与流速无关。蛋白质和肽消化混合物的离线和在线分析均在小于10 fmol的样品水平下进行。HPLC参数可以通过调节洗脱峰宽度来优化，以用于快速LC/MS分析或增强LC/MS/MS实验中的性能。此外，当选定的组分通过界面时，可以大大减少流量，以延长收集质谱数据的可用时间。降低的光谱背景和峰宽操作有助于使用实时自动化仪器控制程序获得肽产物离子光谱（MS/MS）。
A microcapillary liquid chromatography (HPLC) system designed for the gradient elution of peptide and protein samples at now rates < 1 mu L/min has been coupled to a triple-sector quadrupole mass spectrometer via a simple sheathless electrospray interface (microspray). The microspray interface used a name-drawn, uncoated, fused silica needle with tip outer diameters in the range of 15-20 mu m and an opening less than 5 mu m in diameter. Online sample filtration to prevent clogging of the drawn needle was accomplished by using a hydrophilic PVDF membrane filter integrated into the needle assembly. The spray potential (0.5-1 kV) was applied directly to the sample stream through the capillary union, Stable electrospray conditions were obtained over the full range of the gradient (0-90% acetonitrile in water) and was generally independent of flow rate. Both off-line and online analyses of proteins and peptide digest mixtures were performed at sample levels less than 10 fmol. HPLC parameters could be optimized for either rapid LC/MS analysis or enhanced performance in LC/MS/MS experiments by modulation of the eluting peak widths. Additionally, flow could be greatly reduced as selected components pass through the interface to prolong the time available to collect mass spectral data. The reduced spectral background and peak width manipulation facilitated the acquisition of peptide product ion spectra (MS/MS) using real-time, automated instrument control procedures.