Journal of Chromatography A, 1149 (2007) 169–177 Restricted-access material-based high-molecular-weight protein depletion coupled on-line with nano-liquid chromatography–mass spectrometry for proteomics applications Laurent Rieux a , Rainer Bischoff b , Elisabeth Verpoorte a,∗ , Harm A.G. Niederl¨ ander a a Pharmaceutical Analysis Group, University Centre for Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands b Analytical Biochemistry Group, University Centre for Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands Received 16 August 2006; received in revised form 22 January 2007; accepted 23 February 2007 Available online 19 March 2007 Abstract 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 M). 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-m-I.D. reversed-phase trap column are coupled in forward-flush mode to remove albumin before on-line separation on a 50 m I.D. reversed-phase capillary analytical column. Volumes up to 100 L of a complex matrix (containing 0.4 or 4.0 M 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Restricted-access materials; RAM; NanoLC–MS; On-line sample pre-treatment; Proteomics 1. Introduction Liquid chromatography–mass spectrometry (LC–MS) is an important analysis technique in proteomics. In order to increase the concentration sensitivity of LC–MS for low-molecular- weight peptides, it is often necessary to enrich them prior to analysis and to remove high-abundance proteins like albumin. Notably body fluids like serum [1] or cerebrospinal fluid (CSF) [2] often contain considerable amounts of albumin, making the analysis of constituents at nanomolar concentrations or below difficult. Removal of albumin and other high-abundance pro- teins is often achieved by immunoaffinity chromatography based on a panel of immobilised antibodies [1–3]. However, highly abundant proteins can bind lower-abundance proteins or pep- ∗ Corresponding author. Tel.: +31 503633337. E-mail address: E.M.J.Verpoorte@rug.nl (E. Verpoorte). tides, which leads to the loss of the less-abundant species upon removal of the high-abundance species. This is especially true when non-denaturing conditions have to be used as in the case of immunoaffinity chromatography [4]. Consequently, improved methodology based on efficient sample pre-treatment is often necessary to study the low-abundance peptides in complex bio- logical samples containing albumin. Restricted-access materials (RAM) are porous silica materials used in chromatography for the separation of low-molecular-weight analytes from matrix components like albumin by a combination of size exclusion and conventional adsorptive chromatography. Though the applicability of RAM for the analysis of peptides has been demonstrated [5], RAM chromatography is seldom used in proteomics [6]. It has been applied to the analysis of cyanobacterial peptides [7] and low- mass proteins expressed in human fibroblast cells [8] after off-line sample preparation, as well as for the quantitation of neuropeptide Y in porcine plasma. In the latter case, the RAM 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.02.118