Derivatized Cellulose Combined with MALDI-TOF MS: A New Tool for Serum Protein Profiling Isabel Feuerstein, † Matthias Rainer, † Katussevani Bernardo, ‡ Gu 1 nther Stecher, † Christian W. Huck, † Kurt Kofler, § Alexandre Pelzer, § Wolfgang Horninger, § Helmut Klocker, § Georg Bartsch, § and Gu 1 nther K. Bonn* ,† Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, A-6020 Innsbruck, Austria, Biocrates Life Sciences GmbH, Innrain 66, A-6020 Innsbruck, Austria, and Department of Urology, Medical University of Innsbruck, Anichstrae 35, A-6020 Innsbruck, Austria Received July 21, 2005 Providing a rapid and sensitive protein profiling method for biomarker discovery from a variety of biological samples is crucial for the introduction of new markers that improve cancer patient diagnosis at early tumor stages, thus increasing the chances of curative treatment. We report here the development and application of derivatized cellulose particles for selective serum protein profiling. For immobilized metal ion affinity chromatography (IMAC), cellulose was derivatized with glycidyl methacrylate (GMA) and subsequently with iminodiacetic acid (IDA). To investigate the application of this material for generating protein profiles of human serum samples, the serum samples were agitated with the derivatized cellulose particles to a suspension and incubated for 2 h at 30 °C. After washing, 1 µL of the IDA-Cu 2+ -cellulose suspension was applied directly onto a MALDI-target, mixed with sinapinic acid (SA) and analyzed with MALDI-TOF MS. Consistent serum specific data were obtained from aliquoted samples analyzed several times, indicating the reliability of the method. However, the serum fingerprints obtained proved to be specific for any given serum. The technique presented allows a high enrichment of sample on the developed target leading to a high sensitivity and reproducibility without depletion of albumin and immunoglobulin, and sample elution prior to MS-analysis. The study demonstrates for the first time that derivatized cellulose particles combined with MALDI-TOF MS represent a simple, economical, and rapid approach to generate serum protein profiles for biomarker identification. Keywords: spherical cellulose • protein profiling • direct MALDI-TOF • prostate cancer Introduction In many cases, cancer can be cured when detected at an early, organ-confined stage, and there are considerable efforts do develop new biomarkers that improve current diagnosis and prognosis methods for cancer diseases. The identification and analysis of proteins associated with disease is a major challenge. Although several biomarkers for tumor diseases have been identified and introduced successfully into clinical practice, like the prostate-specific antigen (PSA), the carcinoembryonic antigen (CEA), or the alpha-fetoprotein (AFP), their sensitivity and specificity is limited. A good example is prostate cancer, the most frequently diagnosed cancer and the second leading cause of cancer death in men in Western countries. 1 The prostate marker PSA is quite sensitive, however, it does not correctly differentiate benign from malignant prostate disease, and can miss some significant prostate cancers. 2-4 It is war- ranted to search for additional biomarkers in order to improve cancer specificity. Most likely, multiple biomarkers will be required to improve early detection, diagnosis, and prognosis. The classical technique for discovering disease-associated proteins is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by the detection and identification of multiple protein species by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). 5-9 This technique is unchallenged in its ability to resolve thousands of proteins, but it is laborious, requiring large quantities of protein, lacking critical reproducibility standards, and lacking the ability easily convert the results into a routinely used diagnostic test. Therefore, more timesaving and robust techniques are needed. One technique is the ProteinChip approach produced by Ciphergen Biosystems Inc. (Fremont, CA). This method uses surface enhanced laser desorption/ ionization (SELDI) TOF-MS to detect proteins affinity-bound to a protein chip array. 10,11 There have been many examples of the use of SELDI for the determination of disease biomarkers, with the primary focus being diagnostics for all forms of cancer. 12-14 Compared to conventional MS-applications, the SELDI-technology is much easier and timesaving regarding * To whom correspondence should be addressed. Tel.: ++43/(0)512/507- 5171. Fax: ++43/(0)512/507-2943. E-mail: Guenther.Bonn@uibk.ac.at. † Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University. ‡ Biocrates Life Sciences GmbH. § Department of Urology, Medical University of Innsbruck. 2320 Journal of Proteome Research 2005, 4, 2320-2326 10.1021/pr050227z CCC: $30.25  2005 American Chemical Society Published on Web 11/18/2005