Sample Variability of Serum “Albuminome” MALDI Signature for Early Diagnosis of Hepatocellular Carcinoma (HCC): Comparison of Different Methods of Preparation.

SAMPLE VARIABILITY OF SERUM “ALBUMINOME” MALDI SIGNATURES FOR EARLY DIAGNOSIS OF HEPATOCELLULAR CARCINOMA (HCC): COMPARISON OF DIFFERENT METHODS OF PREPARATION Bruschi P.1, Camaggi C.M.1, Cantù M.2, Eletto D.2-3, Strocchi E.1, Genovese F.2, Gramantieri L.5, Chieco P.4, Bolondi L.5 1 Department of Organic Chemistry “A. Mangini”, University of Bologna, Bologna, Italy 2 Proteomic Core Facility, CIRB-CRBA-University Hospital S. Orsola Malpighi, University of Bologna, Bologna, Italy 3 Department of Pharmaceutical Sciences, University of Salerno, Fisciano (SA), Italy (actual affiliation) 4 Applied Biomedical Research Centre (CRBA), University Hospital S. Orsola Malpighi, Bologna, Italy 5 Department of Internal Medicine, University of Bologna, Bologna, Italy E-mail: marco.cantu@unibo.it Background. The serum proteome contains information that directly reflects pathophysiological states. In particular, tumor cells and the surrounding environment might generate peptides of different type and/or at different concentrations than normal cells in a “normal” environment. This “fingerprint” can be detected and compared with controls to identify cancer-specific changes. Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related death worldwide, and an earlier HCC detection improves the percentage of treatable patients. In the present work, we consider if HCC could be diagnosed and staged by analyzing a specific serum “subproteome”, the subset of low molecular weight (LMW) proteins or peptides bound to Human Serum Albumin (HSA), hereafter referred to as “albuminome”. The majority of LMW peptides form complexes with high abundant serum proteins, like HSA, which preserve the bound molecules from clearance and allow them to remain in circulation. Published MALDI-TOF (and SELDI-TOF) procedures are often criticized for the lack of data about analytical reproducibility and the use of inadequate data processing procedures. This study aims to compare the reproducibility of MALDI-TOF data obtained from two different “albuminome” preparation approaches. Methods. In the first method, segregation of albumin was conducted with DakoCytomation® anti-HSA polyclonal antibody coated SiMAG-Cyanuric® paramagnetic silica-based particles. After purification samples were mixed with CHCA matrix and spotted on MALDI target. Acquisition of spectra was performed with an Applied Biosystems® Voyager-DE PRO® instrument. All experiments were carried out in positive, linear ion mode, in evaluating two different mass range, namely from 2500 to 5000 and from 5000 to 15000 Daltons. Preprocessing of raw spectra was performed using in-house developed MATLAB® procedures. In the second method samples underwent affinity column enrichment method, with Calbiochem® ProteoExtract® Albumin Removal Kit. After denaturation and desalting samples were mixed with CHCA matrix and eluted on the MALDI target. MALDI-TOF MS analysis was performed with the same Applied Biosystems® Voyager-DE PRO® instrument in the mass range of 2000 to 20000 Da. Preprocessing of raw data was performed using both the SpecAlign© (Cartwright Group®, University of Oxford) program and our MATLAB procedures. As no generally accepted measure of reproducibility of MALDI-TOF spectra is available, we report here data describing the “mean” coefficient of variation (CV) of the ion intensities. Mean CV was computed as the slope of the regression line correlating mean peak intensity and the corresponding standard deviation, for each peak, under predetermined experimental procedures. The following intra-subject coefficients of variation were computed: intra-sample CV, computed as the mean CV of ion intensities in a single MALDI-TOF run were a standard serum sample was spotted in different positions; intra-day CV, computed as the mean CV observed in independent MALDI-TOF runs carried out on the same sample in the same day; inter-day CV: computed as the mean CV observed in independent MALDI-TO...