Original article Discovery of diagnostic biomarkers for pancreatic cancer in immunodepleted serum by SELDI-TOF MS Aiqun Xue, Robert C. Gandy, Liping Chung, Robert C. Baxter, Ross C. Smith * Department of Surgery, The University of Sydney, Suite 5 Level 5 North Shore Private Hospital, St Leonards, NSW 2065 Australia Keywords: Pancreatic neoplasms Biomarkers Serum proteome Immunodepletion Diagnosis abstract Motivation: Reports of serum pancreatic cancer (PC) biomarkers using SELDI-TOF MS have been incon- sistent because different chip surfaces and interference with high-abundant proteins. This study examines the influence of these factors on the detection of discriminating diagnostic biomarkers. Methods: Serum from fourteen from patients with PC, disease controls (DC, n ¼ 14) and healthy volunteers (HV, n ¼ 14) were evaluated by SELDI using H50, IMAC, Q10 and CM10 chips. A further evaluation was undertaken after depletion of seven high-abundant proteins using spin cartridges. Results: More protein peaks were detected in whole serum than in depleted serum for IMAC, H50 and Q10 chips: 60 vs 39, 56 vs 48 and 69 vs 65, respectively, while the CM10 found less peaks in serum (27 vs 47 peaks). However, there were more differentially expressed peaks in the depleted serum samples for PC vs DC and PC vs HV samples using the H50, Q10 and CM10 ProteinChip arrays, whereas for IMAC arrays, more discriminating peaks were seen in non-depleted serum. The highly significant peaks observed on Q10, CM10 and H50 are consistent with the previous finding of ApoA-I (m/z 27,910e28000) and ApoA-II (m/z 8758 and 17,240). In addition, a number of new discriminating protein peaks were found on different ProteinChip arrays, notably peaks at m/z 4280 and 7763 on IMAC arrays. Conclusion: This study confirms the diagnostic value of ApoA-I&II and identifies further potential diag- nostic biomarkers for pancreatic cancer when multiple chip surfaces are used with depletion of the most highly-abundant proteins. Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved. 1. Introduction The serum of cancer patients contains many proteins which interact with the cancer raising the hope of discovering new biomarkers to aid earlier diagnosis of pancreatic cancer (Rosty C, 2002, 1868; Koopmann J, 2004, 860; Yu Y, 2005, 79, Honda K, 2005, 10,613) [1e4]. Ciphergen Biosystems (Fremont, CA, USA) has developed ProteinChip technology that utilizes Surface-enhanced laser desorption and ionization time of flight mass spectrometry (SELDI-TOP-MS) to facilitate protein profiling of complex biological specimens (Conrade TP, 2004, 163) [5]. It allows the analysis of serum sample sets to discover proteins whose concentrations are influenced by disease states. Recent studies have demonstrated that the detection of pancreatic cancer will probably benefit from a screen of many individual proteins to improve the positive and negative predictive value of CA19.9. Five studies have identified a number of markers (Ehmann, 2007, 205; Takano, 2008; Guo J, 2009, 2292; Fiedler GM, 2009, 3812; Xue A, 2010, 391) [6e10] which appear to discriminate serum from pancreatic cancer cases and disease control groups and from healthy volunteers. These studies demonstrated different protein markers but they have used different chip surfaces and therefore different methods of selection. Another important variable is the method of preparation of the serum sample. Some studies have examined undepleted serum while other studies filter off the high-abundant proteins such as albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin and fibrin- ogen because they may conceal important low-abundance proteins. These variances in technique and patients could lead to apparent non-reproducible results between studies [11]. Different protein groups would be expected to adhere to the different chip surfaces: The ProteinChip H50 array binds proteins through reverse-phase or hydrophobic interaction chromatography; the CM10 ProteinChip arrays incorporate a carboxylate chemistry (negatively charge) and thus acts as a weak cationic exchanger, while the ProteinChip Q10 array incorporates quaternized ammo- nium groups (positively charged) and thus acts as a strong anion * Corresponding author. Tel.: þ61 2 94373511; fax: þ61 2 94373522. E-mail address: ross.smith@sydney.edu.au (R.C. Smith). Contents lists available at SciVerse ScienceDirect Pancreatology journal homepage: www.elsevier.com/locate/pan 1424-3903/$ e see front matter Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved. doi:10.1016/j.pan.2012.02.009 Pancreatology 12 (2012) 124e129