Molecular Psychiatry (2010) 15, 1088–1100 & 2010 Macmillan Publishers Limited All rights reserved 1359-4184/10 www.nature.com/mp ORIGINAL ARTICLE Global proteomic profiling reveals altered proteomic signature in schizophrenia serum Y Levin1,3, L Wang1,3, E Schwarz1, D Koethe2, FM Leweke2 and S Bahn1 1Institute of Biotechnology, University of Cambridge, Cambridge, UK and 2Department of Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany Schizophrenia is one of the most severe psychiatric disorders affecting 1% of the world population. There is yet no empirical method to validate the diagnosis of the disease. The identification of an underlying molecular alteration could lead to an improved disease understanding and may yield an objective panel of biomarkers to aid in the diagnosis of this devastating disease. Presented is the largest reported liquid chromatography-mass spectro- metry-based proteomic profiling study investigating serum samples taken from first-onset drug-naive patients compared with samples collected from healthy volunteers. The results of this large-scale study are presented along with enzyme-linked immunosorbent assay-based validation data. Molecular Psychiatry (2010) 15, 1088–1100; doi:10.1038/mp.2009.54; published online 23 June 2009 Keywords: schizophrenia; biomarkers; label free; nanoLC-MS/MS; LC-MSE; serum Introduction techniques enable the measurement of disease- associated changes in a large number of proteins Schizophrenia is a severe, complex neuropsychiatric simultaneously. disorder. Despite substantial variation, especially Proteomic studies have many advantages over with regard to gender, urbanicity and migrant status, mRNA gene expression analyses as they focus on schizophrenia affects approximately 1% of the popu- the protein as the ‘biological effector molecule’.6 lation, on a global scale.1,2 Most proteomics platforms are centred around the Currently, there is no empirical test aiding the implementation of mass spectrometry techniques in accurate diagnosis of people suffering from schizo- conjunction with separation technologies.5 Over the phrenia. Although some insights into the aetiology of past decade, these technologies have matured and schizophrenia have been gained using the current now enable the relative abundance of measurements interview-based methodology, the molecular basis of of hundreds or even thousands of proteins in one the disease is still unknown. A more biomedical experiment.5 Thus, researchers can monitor the global approach to understanding the aetiology of the expression of proteins and protein groups in search disease would improve management and treatment for disease-related differences, which can provide an of schizophrenia. insight into the aetiology of diseases and the ability to Several studies have been conducted in search for find disease-specific markers. genetic mutations linked to the disorder. However, no One of the major requirements of an objective genetic polymorphism is consistent across the differ- biomedical diagnostic test is that the biological ent studies.3 Genetic factors alone do not account for sample must be accessible. However, this is not the illness onset,4 which is heavily influenced by envir- case for every body fluid or tissue. Central nervous onmental factors. system tissue, for example, is clinically not readily The proteome holds the key to unravelling disease accessible for analysis. In the case of neuropsychiatric mechanisms enabling the development of imp- disorders, this is a critical issue, as these disorders roved diagnostics, the assessment of drug response, manifest themselves with brain dysfunctions. Serum, drug efficacy and drug toxicity.5 The latest proteomic on the other hand, is a highly accessible bodily fluid, which can be sampled with minimal discomfort to the Correspondence: Dr S Bahn, Institute of Biotechnology, Univer- patient. Moreover, it has been shown that for many sity of Cambridge, Tennis Court Road, Cambridge, Cambridge- diseases, the serum proteome holds the greatest shire CB 2 1QT, UK. promise for the discovery of disease biomarkers.7 E-mail: [email protected] 3These authors contributed equally to this work. One of the main reasons for this is that it circulates Received 27 January 2009; revised 7 April 2009; accepted 4 May and is in molecular exchange with every tissue and 2009; published online 23 June 2009 every organ in the body. It therefore reflects both Altered proteomic signature in schizophrenia serum Y Levin et al 1089 physiological and pathological processes in the Table 1 Demographic information for the clinical serum body and is most suited for disease diagnosis and samples used in the profiling study (values are mean±s.d.) monitoring. Analysis of the serum proteome, however, holds Schizophrenia Control Significance several challenges for quantitative proteomics. The first and the foremost is the overwhelming dynamic Serum sample size n =22 n =33 ± ± range of abundance of the proteins. It is estimated that Age 29.0 11 28 7 P = 0.71 Gender (male/female) 15/7 18/15 P = 0.31 it spans across 10–12 orders of magnitude.8 This is an enormous challenge as most high-throughput quanti- tative platforms only have a dynamic range of up to 3 orders of magnitude.9 Another challenge of the analysis of the serum is the diverse range of analytes Sample preparation present. These include very large molecules such as All samples were prepared and analysed blindly and proteins, and smaller molecules such as lipids and randomly. The total protein concentration of each other metabolites as well as electrolytes.7 Finally, it is serum sample was measured using a protein assay estimated that serum includes around 10 000 indivi- (Bio-Rad, Hercules, CA, USA) before preparation. dual proteins.7,8 These challenges must be dealt with Each sample was depleted of the 20 most abundant during sample preparation to extract maximum proteins: albumin, immunoglobulin (Ig)G, IgA, IgM, information from the precious clinical samples. IgD, transferrin, fibrinogen, a2-macroglobulin, a1- To overcome the aforementioned challenges, we antitrypsin, haptoglobulin, a1-acid glycoprotein, cer- used a non-hypothesis-based label-free proteomic uloplasmin, apolipoprotein A1, apolipoprotein A2, approach that is sensitive, reproducible and accurate apolipoprotein B, complement C1q, complement C3, as described previously in Levin and Schwarz.10 The complement C4, plasminogen and transthyretin. This presented study is the largest liquid chromatography- was performed using an immunoaffinity kit (Sigma, mass spectrometry (LC-MS)-based proteomic profil- St Louis, MO, USA), loading a total of 560 mgof ing study conducted to date for the purpose of protein (average of 5 ml) of each sample onto the investigating sera taken from first-onset drug-naive depletion column. Buffer exchange was performed schizophrenia patients. with 50 mM ammonium bicarbonate using spin columns (Millipore, Bedford, MA, USA) with a 5- kDa-molecular weight cutoff to separate proteins from Materials and methods small molecules in the serum. The proteins were reduced using 5 mM dithriotheitol (Sigma, USA) at The Ethical Committee of the Medical Faculty of the 60 1C for 30 min and alkylated with 10 mM iodoacete- University of Cologne reviewed and approved the mide (Sigma, USA) in the dark at room temperature protocol of this study and the procedures for sample for 30 min. The proteins were digested using trypsin collection and analysis. All study participants gave (Promega, Madison, WI, USA), at a ratio of 1:50 (w/w their written informed consent. All clinical investiga- trypsin/protein) for 16 h at 37 1C. The digestion was tions were conducted according to the principles stopped by adding 2.3 ml of 8.8 M HCl to each sample. expressed in the Declaration of Helsinki. The samples were stored at À80 1C until analysis. Before nanoUPLC-MSE analysis, each sample was Clinical serum samples spiked with 25 fmol mlÀ1 tryptically digested Enolase A set of 55 clinical samples were prepared. The from Yeast (Waters, Milford, MA, USA), which was samples have been stored at À80 1C in identical used for normalization. Pure Saccharomyces cerevi- conditions following collection and have undergone siae Enolase digest was injected with identical LC and two freeze–thaw cycles. The samples included 22 MS methods to assure correct peptides were selected serum samples from patients diagnosed with first- for normalization (data not shown). onset paranoid schizophrenia (DSM-IV 295.30) and 33 serum samples taken from demographically Liquid chromatography matched controls. The healthy volunteers were For all chromatographic steps, LC-MS grade solvents chosen carefully to make sure that there was no were used (Fisher Scientific, Loughborough, UK). family history of Schizophrenia or other detectable Each sample was injected and analysed three times psychiatric, neurological or medical history. Lastly, followed by a blank injection (to ensure there is no the healthy volunteers were demographically carry-over of peptides from one sample to the other in matched to the diseased patients (see Table 1). this sequential process), except for the QC samples that were injected once. For each sample, approxi- Quality control samples mately 0.5 mg of total protein digest was loaded using Along with the 55 clinical samples were 12 quality split-less nano-Ultra Performance Liquid Chromato- controls (QCs) that were aliquoted from one serum graphy (10kpsi nanoAcquity; Waters). Buffers used sample before any preparation.