Elicone C, Lui M, Geromanos S, Erdjument-Bromage H, Tempst P
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
J Chromatogr A. 1994 Jul 29;676(1):121-37. doi: 10.1016/0021-9673(94)00089-1.
An optimized microbore RP-HPLC system (1.0 mm I.D. columns) for the purification of low picomole amounts (< 5 pmol) of peptides is described. It is comprised of commercially available columns, instrument components and parts. These were selected on the basis of a comparative evaluation and to yield the highest resolution and most efficient peak collection. The sensitivity of this system equals, probably surpasses, that of advanced chemical microsequencing for which 2-4 pmol of peptide are minimally required. As an automated sequencer cannot be "on-line" connected with a micro-preparative HPLC system, fractions must be collected and transferred. With a typical flow of 30 microliters, efficient manual collection is possible and fractions (about 20 microliters in volume) can still be handled without unacceptable losses, albeit with great precaution. Furthermore, major difficulties were encountered to efficiently and quantitatively load low- or sub-picomole amounts of peptide mixtures onto the RP-HPLC column for separation. Discipline and rigorous adherence to sample handling protocols are thus on order when working at those levels of sensitivity. With adequate instrumentation and handling procedures in place, we demonstrate that low picomole amounts of peptides can now be routinely prepared for analysis by combined Edman-chemical sequencing-matrix-assisted laser-desorption mass spectrometry (MALDI-MS). The integrated method was applied to covalent structural characterization of minute quantities of a gel-purified protein of known biological function but unknown identity. The results allowed unambiguous identification and illustrated the power of MALDI-MS-aided interpretation of chemical sequencing data: accurate peptide masses were crucial for (i) confirmation of the results, (ii) deconvolution of mixed sequences, (iii) proposal of complete structures on the basis of partial sequences, and (iv) confirmation of protein identification (obtained by database search with a single, small stretch of peptide sequence) by "mass matching" of several more peptides with predicted proteolytic fragments.
本文描述了一种优化的微径反相高效液相色谱（RP-HPLC）系统（内径1.0 mm的色谱柱），用于纯化低皮摩尔量（<5 pmol）的肽段。该系统由市售的色谱柱、仪器组件和部件组成。这些组件是在比较评估的基础上选择的，以实现最高的分辨率和最有效的峰收集。该系统的灵敏度等同于，甚至可能超过先进的化学微测序技术，后者至少需要2-4 pmol的肽段。由于自动测序仪无法与微制备HPLC系统“在线”连接，因此必须收集并转移馏分。在典型流速为30微升的情况下，可以进行有效的手动收集，并且尽管需要格外小心，但体积约为20微升的馏分仍可处理，且损失在可接受范围内。此外，将低皮摩尔量或亚皮摩尔量的肽混合物有效且定量地加载到RP-HPLC柱上进行分离时遇到了重大困难。因此，在如此低的灵敏度水平下工作时，必须遵守规范并严格遵循样品处理方案。通过适当的仪器和处理程序，我们证明现在可以常规制备低皮摩尔量的肽段，用于通过埃德曼化学测序-基质辅助激光解吸质谱（MALDI-MS）联用进行分析。该综合方法应用于对微量凝胶纯化的、具有已知生物学功能但身份未知的蛋白质进行共价结构表征。结果实现了明确的鉴定，并展示了MALDI-MS辅助化学测序数据解释的强大功能：准确的肽质量对于（i）结果确认、（ii）混合序列的解卷积、（iii）基于部分序列提出完整结构以及（iv）通过将更多肽段与预测的蛋白水解片段进行“质量匹配”来确认（通过使用一小段肽序列在数据库中搜索获得的）蛋白质鉴定至关重要。
