Wang Hong, Hanash Samir M
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
J Proteome Res. 2008 Jul;7(7):2743-55. doi: 10.1021/pr700876g. Epub 2008 May 31.
We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 microm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet > or = 0.9, and peptides matched > or = 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events.
我们报道了一种全新设计的全自动、高效并行非分流纳流毛细管HPLC系统，该系统与线性离子阱（LTQ）和高性能纳电喷雾电离傅里叶变换离子回旋共振质谱（nanoESI LTQ-FTICR MS）在线联用。该系统旨在对复杂蛋白质混合物（尤其是血清和血浆）进行高通量蛋白质组分析，它由两根反相捕集柱组成，用于大体积进样、高速进样加载和脱盐，还有两根反相分析毛细管柱。通过一个纳米级两位十通切换阀，整个系统连接到内径为10微米的纳米发射器，该发射器安装在LTQ-FTICR MS采样锥前的纳电喷雾源上。单个HPLC系统通过两个独立的二元梯度泵系统同时向两根纳流速率毛细管柱提供梯度洗脱。并行毛细管柱方法消除了柱再生/平衡的时间延迟，因为一根毛细管柱用于分离样品混合物并将分离的馏分输送到质谱仪，而另一根毛细管柱正在进行再生和平衡。目前这种自动化并行纳流速率毛细管HPLC系统的保留时间和峰强度的重现性与使用单柱配置获得的重现性相当。对胰蛋白酶消化产物的重复进样表明，该系统在两根柱上都具有良好的保留时间和峰面积重现性，平均CV值分别小于1.08%和7.04%。与单毛细管柱LC-MS流程相比，在2小时的LC-MS分析中，通量提高到了100%。该系统在血浆蛋白质组学研究中的应用得到了证明。总共采集了312868次MS/MS事件，高可信度鉴定出1564种蛋白质（Protein Prophet≥0.9，且匹配肽段≥2）。使用单柱LC-MS与并行柱LC-MS运行一系列血浆组分的比较表明，并行柱LC-MS系统显著减少了样品残留，提高了质谱数据质量，并增加了MS/MS序列扫描事件的数量。
