Multidimensional liquid chromatography is the mainstay separation technique used for shotgun proteomic analyses. The application of a multiple‐fraction concatenation (MFC) strategy can result in a more disperse and consistent peptide elution profile across different fractions, when compared with a conventional strategy. Herein, we present the first automated online RP‐RP platform implementing an MFC strategy to facilitate robust, unattended, routine proteomic analyses. The improved duty cycle utilization of the MFC strategy led to an increase of 9% in the separation space occupancy and increases of approximately 10% in the identification of both proteins and peptides. The peptides uniquely identified by the MFC strategy were significantly biased toward those of acidic nature, with increased precursor signals leading to improved MS/MS spectral quality and enhanced acidic peptide identification. These improvements in qualitative analysis using the MFC strategy were also extended to quantitative analysis. When the acquired proteome was quantified with a normalized spectral abundance factor, the additionally acquired acidic peptides were a critical factor leading to enhanced reproducibility of quantitation using the MFC strategy. With merits of superior qualitative and quantitative characteristics over the conventional strategy, the MFC strategy appears to be a highly amenable technique for enhancing the separation capacity for routine proteomic analyses.

中文翻译：

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在线二维高/低pH反相/反相液相色谱平台中多级联策略的实施，用于定性和定量shot弹枪蛋白质组分析

多维液相色谱是用于shot弹枪蛋白质组分析的主要分离技术。与常规策略相比，多级联（MFC）策略的应用可以在不同馏分之间产生更分散，更一致的肽洗脱图。本文中，我们介绍了第一个实现MFC策略的自动化在线RP-RP平台，以促进可靠，无人值守的常规蛋白质组学分析。MFC策略提高了占空比利用率，导致分离空间占用率增加了9％，而蛋白质和多肽的鉴定则增加了约10％。通过MFC策略唯一识别的肽明显偏向于酸性肽，具有增加的前体信号，从而改善了MS / MS光谱质量并增强了酸性肽的鉴定 使用MFC策略在定性分析中的这些改进也扩展到了定量分析。当用标准化的光谱丰度因子对获得的蛋白质组进行定量时，另外获得的酸性肽是导致使用MFC策略提高定量重现性的关键因素。MFC策略具有优于常规策略的定性和定量特性，因此它是一种用于常规蛋白质组分析的增强分离能力的高度易用的技术。当用标准化的光谱丰度因子对获得的蛋白质组进行定量时，另外获得的酸性肽是导致使用MFC策略提高定量重现性的关键因素。MFC策略具有优于常规策略的定性和定量特性，因此它是一种用于常规蛋白质组分析的增强分离能力的高度易用的技术。当用标准化的光谱丰度因子对获得的蛋白质组进行定量时，另外获得的酸性肽是导致使用MFC策略提高定量重现性的关键因素。MFC策略具有优于常规策略的定性和定量特性，因此它是一种用于常规蛋白质组分析的增强分离能力的高度易用的技术。