LABEL-FREE QUANTIFICATION VIA IMPROVED CHROMATOGRAPHIC ELUTION REPRODUCIBILITY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 相对肽定量的最直接和直观的方法是比较不同实验中相同肽物质的峰面积。 然而，确定属于相同肽物质的峰的困难来自于通过HPLC的肽保留时间的再现性差。 在MudPIT实验期间，内部填充微柱与色谱分离的第二维的组合使用进一步降低了保留再现性。 我们提出了一种色谱方法，该方法利用了大1 mm i.d. RP柱，并将其与纳米喷雾ESI的灵敏度相结合。 使用大容量1.0x30.0 mm RP色谱柱，通过长单相梯度进行色谱洗脱。 高线性速度流（2 ml/min）在柱后分流，其中样品以250 nl/min的纳米喷雾方案引入仪器。这种设置改善了色谱重现性，并提高了检测灵敏度，这与毛细管微柱纳米ESI设置相似

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The most direct and intuitive method of relative peptide quantification is comparison of the peak areas of the same peptide specie across different experiments. However, the difficulty of determining peaks that belong to the same peptide specie comes from poor reproducibility of the peptide retention time via HPLC. Combined use of the in-house packed micro columns with a second dimension of chromatographic separation during MudPIT experiments reduces retention reproducibility even further. We propose a chromatographic approach that takes advantage of the high retention reproducibility of a large 1mm i.d. RP column and combines it with the sensitivity of the nano-spray ESI. Chromatographic elution is carried out via long single phase gradients using large capacity 1.0x30.0 mm RP column. The high linear velocity flow (2ml/min) is split post column where the sample is introduced into the instrument at the nano-spray regime of 250nl/min. This setup accounts for improved chromatographic reproducibility, and increased detection sensitivity that is similar to the capillary micro-column nano-ESI setup