Abstract：
Peptide separations that combine high sensitivity, robustness, peak capacity, and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with offline gradient generation. One binary pump generates gradients in an accelerated fashion to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshooting. A high degree of parallelization is possible, as one sample undergoes separation while the next sample plus its corresponding mobile phase gradient are transferred into the storage loop and a third sample is loaded into a sample loop. Selective offline elution from the trap column into the sample loop prevents salts and hydrophobic species from entering the analytical column, thus greatly enhancing column lifetime and system robustness. With this design, samples can be analyzed as fast as every 20 min at a flow rate of just 40 nL/min with close to 100% MS utilization time and continuously for as long as several months without column replacement. We utilized the system to analyze the proteomes of single cells from a multiple myeloma cell line upon treatment with the immunomodulatory imide drug lenalidomide.
摘要：
结合高灵敏度的肽分离，鲁棒性，峰值容量，和通量对于将自下而上的蛋白质组学扩展到包括单细胞在内的较小样品至关重要。为此,我们开发了一种具有离线梯度生成的多柱纳米LC系统。一个二元泵以加速的方式生成梯度，以支持多个分析柱，和一个单一的陷阱柱接口与所有分析柱，以减少所需的维护和简化故障排除。高度并行化是可能的，例如,一个样品经历分离,而下一个样品加上其相应的流动相梯度被转移到储存回路中,并且第三样品被加载到样品回路中。从捕集柱到样品回路的选择性离线洗脱可防止盐和疏水性物质进入分析柱，从而大大提高列的寿命和系统的鲁棒性。有了这个设计，样品可以以每20分钟的速度进行分析，流速仅为40nL/min，使用时间接近100％MS，连续长达数月，无需更换色谱柱。我们利用该系统分析了用免疫调节酰亚胺药物来那度胺治疗的多发性骨髓瘤细胞系中单细胞的蛋白质组。
