LABEL-FREE QUANTIFICATION VIA IMPROVED CHROMATOGRAPHIC ELUTION REPRODUCIBILITY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 相对多肽定量的最直接和最直观的方法是通过不同的实验比较同一多肽物种的峰面积。然而，很难确定属于同一多肽物种的峰，这是因为通过高效液相色谱法测定多肽保留时间的重现性较差。在MudPIT实验期间，将内部填充的微柱与二维色谱分离结合使用，进一步降低了保留重现性。我们提出了一种利用大的1 mm内径的高保留重现性的层析方法。RP柱，并将其与纳米喷雾ESI的灵敏度相结合。色谱柱为大容量1.0x30.0 mm反相柱，采用长单相梯度洗脱。高线速度流(2ml/min)是分离柱子，样品以250nl/min的纳米喷雾制度引入仪器。这种设置可改善色层重现性，并提高检测灵敏度，类似于毛细管微柱纳米ESI设置

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