TECHNICAL BRIEF Automated serum peptide profiling using novel magnetic C18 beads off-line coupled to MALDI-TOF-MS Connie R. Jimenez 1, 2 , Zineb El Filali 1 , Jaco C. Knol 1 , Klaas Hoekman 1 , Frank A. E. Kruyt 1 , Giuseppe Giaccone 1 , August B. Smit 2 and Ka Wan Li 2 1 OncoProteomics Laboratory, Department of Medical Oncology, VU Medical Center, Amsterdam, The Netherlands 2 Department of Molecular and Cellular Neurobiology, Center for Medical Systems Biology/Neurogenomics and Cognitive Research, Vrije Universteit, Amsterdam, The Netherlands Serum peptide profiling by MS is an emerging approach for disease diagnosis and biomarker discovery. A magnetic bead-based method for off-line serum peptide capture coupled to MALDI- TOF-MS has been recently introduced. However, the reagents are not available to the general scientific community. Here, we developed a protocol for serum peptide capture using novel magnetic C18 beads, and automated the procedure on a high-throughput magnetic particle pro- cessor. We investigated bead equilibration, peptide binding and peptide elution conditions. The method is evaluated in terms of peaks counts and reproducibility of ion intensities in control serum. Overall, the DynaBead-RPC18-based serum sample processing protocol reported here is reproducible, robust and allows for the detection of ,200 peptides at m/z 800–4000 of serum that was allowed to clot for 1 h. The average intra-experiment %CV of normalized ion intensities for crude serum and 0.5% TFA/0.15% n-octyl glucoside-treated serum, respectively, were 12% (range 2–38%) and 10% (3–21%) and the inter-experiment %CVs were 24% (10–53%) and 31% (10–59%). Importantly, this method can be used for serum peptide profiling by anyone in pos- session of a MALDI-TOF instrument. In conjunction with the KingFisher  96, the whole serum peptide capture procedure is high-throughput (,20 min per isolation of 96 samples in parallel), thereby facilitating large-scale disease profiling studies. Received: September 22, 2006 Revised: January 12, 2007 Accepted: March 12, 2007 Keywords: Automation / MALDI-TOF-MS / Magnetic beads / Reproducibility / Serum 598 Proteomics Clin. Appl. 2007, 1, 598–604 In the past few years, MS is increasingly used to profile the low molecular weight proteome of serum in a variety of diseases [1, 2]. The general aim is to identify biomarker pat- terns that can be used for diagnosis, prognosis or monitoring of disease. Current efforts in working towards this goal are focused on the development of standard procedures for pre- analytical sample handling and reliable procedures for serum peptide sample processing and profiling. Ultimately, the impact in the field of oncology may be significant, where early diagnosis and therapy-response monitoring can make a significant difference in outcomes. So far, most studies employed TOF mass spectrometers with SELDI, where peptide binding is performed on chro- matographic surfaces [2, 3]. The disadvantages of this approach are the low resolution of the SELDI-TOF instru- ments and the limited binding capacity of a surface. More recently, a few studies have used high-resolution instru- ments with MALDI coupled to a TOF ion detector. These MALDI-TOF-MS studies mainly made use of a magnetic bead-based method for off-line serum peptide capture [4–7]. Unfortunately, these magnetic beads are not generally avail- able [6] or only to those with a mass spectrometer from a Correspondence: Dr. Connie R. Jimenez, OncoProteomics Labo- ratory, Department of Medical Oncology, VU Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands E-mail: c.jimenez@vumc.nl Fax: 131-204443844 DOI 10.1002/prca.200600483  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com