Department of Clinical Chemistry, University Medical Center Hamburg - Eppendorf, Hamburg, Germany.
J Sep Sci. 2010 Nov;33(21):3283-91. doi: 10.1002/jssc.201000474.
The influence of packing process parameters (packing pressure, application of ultrasound) and the stationary phase particle size (3.5 and 5 μm) on the chromatographic performance of HPLC/MS chips was systematically investigated for proteomic samples. First, reproducibility and detection limits of the separation were evaluated with a low-complexity sample of tryptic BSA peptides. The influence of adsorbent packing quality on protein identification was then tested with a typical proteomics sample of high complexity, a human plasma protein fraction (Cohn fraction IV-4). All HPLC/MS chips provided highly reproducible separations of these proteomic samples, but improved packing conditions and smaller particle sizes resulted in chromatograms with narrower peaks and correspondingly higher signal intensities. Improved separation performance increased the peak capacity, the number of identified peptides, and thus the sequence coverage in the proteomic samples, particularly for low sample amounts.
本研究系统考察了装填工艺参数（装填压力、施加超声）和固定相粒径（3.5μm 和 5μm）对 HPLC/MS 芯片在蛋白质组样品分析中的色谱性能的影响。首先，利用低复杂度的胰蛋白酶 BSA 肽样品评估了分离的重现性和检测限。然后，利用高复杂度的典型蛋白质组样品，即人血浆蛋白质分馏物（Cohn 分馏物 IV-4），测试了吸附剂装填质量对蛋白质鉴定的影响。所有 HPLC/MS 芯片均能高度重现地分离这些蛋白质组样品，但改善的装填条件和更小的粒径可使色谱峰变窄，相应的信号强度更高。改善的分离性能提高了峰容量、鉴定肽的数量，从而提高了蛋白质组样品中的序列覆盖率，尤其是对于低样品量的情况。
