Restricted-access material-based high-molecular-weight protein depletion coupled on-line with nano-liquid chromatography–mass spectrometry for proteomics applications

Proteomics samples often contain both abundant proteins and low-level proteins and peptides. Highly abundant proteins can mask and/or bind those of lower abundance and thereby hinder their analysis. In particular, we were concerned with samples containing large amounts of albumin (up to 4.0 μM). In this study, a novel set-up for multidimensional nano-liquid chromatography–mass spectrometry (nanoLC–MS) with three columns coupled on-line was developed and characterised. A 1-mm-I.D. restricted-access-material (RAM) cartridge and a 100-μm-I.D. reversed-phase trap column are coupled in forward-flush mode to remove albumin before on-line separation on a 50 μm I.D. reversed-phase capillary analytical column. Volumes up to 100 μL of a complex matrix (containing 0.4 or 4.0 μM albumin) could be injected onto this system, enabling a 5000-fold volume reduction. Up to 99.7% of the albumin present in samples could be efficiently removed over the RAM cartridge. The total analysis time was about 40 min. Using Substance P as a model peptide, separations were efficient, with a peak width of 10 s at half height. Moreover, separations were highly reproducible (relative standard deviation (RSD) on retention time ∼3% over 1 week). The set-up proved to be robust and was used for about 750 analyses without exchanging one of the columns. Flexibility with respect to the stationary phase material in the sample preparation cartridge allows for other separation modes to be applied as well.