Investigation of carryover of peptides in nano-liquid chromatography/mass spectrometry using packed and monolithic capillary columns

This article relates on reversed-phase column technology as the main cause of carryover in the LC–MS/MS analysis of proteomics samples. The separation performance and column carryover was investigated using four capillary columns with different morphologies by monitoring the remaining traces of tryptic peptides of bovine serum albumin in subsequent blank LC–MS runs. The following trend in column carryover was observed: capillary column packed with 3 μm porous C18 particles ≫ 2.7 μm fused-core C18 packed column > silica C18 monolith ≫ poly(styrene-co-divinylbenzene) monolith. This is mainly related to the intrinsic properties of the different chromatographic materials, related to surface area and the presence and size of mesopores (stagnant zones where mass transfer is controlled by diffusion). Both isocratic and gradient wash steps with 2-propanol/acetonitrile mixtures were not effective to reduce column carryover. An isocratic wash step using a high acetonitrile percentage or blank gradient reduced carryover with approximately 50%. Nevertheless, it is important to note that effects of column carryover were still observed in a fifth subsequent gradient blank. Although the polymer monolith clearly outperformed the silica materials in terms of carryover, this material exhibited also the lowest loadability, which may be a disadvantage when profiling proteomics mixtures with a broad dynamic range.

► Carryover effects is an underestimated phenomenon in proteomic research.
► Analytical column represents the main source of carryover in the HPLC system.
► Carryover effects can still be observed after executing five gradient blanks.
► Capillary column packed with porous particles showed higher effects than monoliths.