Separation optimization of long porous‐layer open‐tubular columns for nano‐LC–MS of limited proteomic samples

The single-run resolving power of current 10 μm id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (∼0.75 μm thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE–PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2σ definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (−/−) cells, and ∼1200 and 2500 proteins in extracts of 10 000 and 100 000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.