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Separation optimization of long porous‐layer open‐tubular columns for nano‐ LC – MS of limited proteomic samples
Author(s) -
Rogeberg Magnus,
Vehus Tore,
Grutle Lene,
Greibrokk Tyge,
Wilson Steven Ray,
Lundanes Elsa
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201300499
Subject(s) - porosity , chemistry , chromatography , monolith , analytical chemistry (journal) , monolithic hplc column , volumetric flow rate , resolution (logic) , nano , nanotechnology , materials science , high performance liquid chromatography , composite material , biochemistry , computer science , physics , quantum mechanics , artificial intelligence , catalysis , organic chemistry
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 B x PC 3 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 W nt signaling pathway.