Comparison of Commercial Nanoliquid Chromatography Columns for Fast, Targeted Mass Spectrometry-Based Proteomics

Abstract

Aim:We compared four commonly used, commercially available reverse phase nanoLC columns for identification/determination of Wnt/β-catenin-related pathway proteins.Materials & methods:The columns were: Chromolith® (silica monolith; Merke Millipore, MA, USA), PepMap™ (porous particles; Thermo Fisher Scientific, MA, USA), Accucore™ (solid core particles; Thermo Fisher Scientific) and PepSwift™ (organic monolith; Thermo Fisher Scientific).Results:The peak capacity of the columns varied from 100 (Pepswift) to 190 (Accucore) (for 30 min gradients). All columns enabled identification/detection of GSK3β and β-catenin in the complex samples. However, even the columns with higher peak capacities could not enable detection of the somewhat less abundant proteins AXIN2 and TNKS2. The monoliths were more prone to retention time instability when sample complexity increased.Conclusion:We find that commercial nanoLC columns, although featuring different morphologies and peak capacities, provided surprisingly few practical differences for relatively fast, targeted determination of proteins.

Versatile, sensitive liquid chromatography mass spectrometry – Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins

“…The cells were lyzed using a standard protocol as described in ref. 44 and protein extracts were loaded onto a 3–8% Bis-Tris Acetate gel (NuPAGE, Invitrogen, CA, USA) and separated at 150 V. The gel was subsequently stained in Coomassie Blue staining solution according to a standard protocol described in Johnsen et al 45…”

“…Peak capacities were measured manually as described in detail in ref. 44. Selection of signature peptides were done with Skyline software46 (v.1.7), peptideATLAS47 and Uniprot48.…”

“…Comprehensive protein identification was done with the SEQUEST algorithm in Thermo Proteomics Discoverer (v.1.4) software using standard search settings as described in detail in ref. 44.…”

Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

“…The study recommend monolithic trap column because it provided significantly reduced peak widths, required fewer connective parts (hence smaller dead volume), provided lower back pressure (enabled fast loading and equilibration), and self‐prepared monolithic trap columns provided the luxury of choosing which functional groups to include. The same research group compared commercial analytical monolithic and particle‐based capillary columns in target quantification of cancer cell proteins involved in a metabolic pathway of interest . The authors could not find significant differences, although slight retention time instability was noticed with monolithic columns.…”

“…Application of DIA on high‐resolution MS systems, five years ago, substantially increased the number of peptides that could be simultaneously quantified, and simplified the design of acquisition methods . Recently, a study was published comparing silica‐C8 based monolithic trap and particle‐based‐C18 trap columns using a targeted approach . The study recommend monolithic trap column because it provided significantly reduced peak widths, required fewer connective parts (hence smaller dead volume), provided lower back pressure (enabled fast loading and equilibration), and self‐prepared monolithic trap columns provided the luxury of choosing which functional groups to include.…”