A fully automated 2‐D LC‐MS method utilizing online continuous pH and RP gradients for global proteome analysis

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Abstract
The conventional 2‐D LC‐MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong‐cation‐exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2‐D LC‐MS/MS system is introduced that provided excellent separation and identification power. The pH gradient‐based elution provided more basic peptides than that of salt‐based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution‐based 2‐D LC‐MS/MS approaches were compared by analyzing the mouse liver proteome.