Plasma Levels of Complement 4a Protein are Increased in Alzheimer’s Disease Stuart Bennett, BSc,* Melissa Grant, PhD,w Andrew J. Creese, PhD,wz Francesca Mangialasche, MD,y Roberta Cecchetti, MLT,y Helen J. Cooper, PhD,z Patrizia Mecocci, MD, PhD,y and Sarah Aldred, PhD* Abstract: Alzheimer’s disease (AD) is a devastating neurodegener- ative disorder that has been predicted to affect 106.2 million people worldwide by 2050. Currently, definitive diagnosis for this disease is given post mortem, and there is a need for biomarker identification to enable earlier diagnosis of this disease. Biomarkers of AD would ideally represent early disease process and will be present in peripheral tissue before cognitive decline develops in this population. Proteomic technologies offer a strategy to under- take such work. In recent times, research in this field has moved away from classical 2-dimensional gel-based proteomics toward more sensitive, non-gel–based proteomic methodologies. In the study presented here, isobaric labeling for relative and absolute quantification was used to assess plasma protein expression in a small group of AD and control samples. Several proteins were identified as being differentially expressed between these 2 populations. Complement 4a plasma protein was identified as increased in AD by isobaric labeling for relative and absolute quantification, and this finding was further validated by Western blotting and enzyme-linked immunosorbent assay. These data suggest that inflammatory processes, which have been shown to be involved in AD pathology in the brain, are also present in plasma. Key Words: Alzheimer’s disease, iTRAQ, proteomics, biomarker, mass spectrometry (Alzheimer Dis Assoc Disord 2012;26:329–334) A lzheimer’s disease (AD) is a neurodegenerative disorder that, alarmingly, has been predicted to affect 106.2 million people worldwide by 2050. 1 This disease is characterized by the presence of extracellular amyloid-b (Ab) peptides and intracellular neurofibrillary tangles comprising hyperphos- phorylated tau in the brain. 2 At present, definitive diagnosis for this disease is given post mortem on the basis of these characteristic neuropathologies. Identification of a biomarker or series of biomarkers would be advantageous to enable earlier diagnosis of this disease, ideally before cognitive decline in this population develops. Early diagnosis would allow directed treatments and facilitate the monitoring of interventions (eg, antioxidant supplementation or exercise). Plasma comprises hundreds of proteins 3,4 and harbors hallmarks of processes and reactions that take place in the body. 5,6 In addition to this, half a liter of cerebrospinal fluid (CSF), which is in direct contact with the extra- cellular space of the brain, and therefore reflects changes that occur in the brain, is converted into plasma on a daily basis. 7–9 Therefore, biochemical changes that are a part of AD brain pathology (eg, inflammation) may also be present in AD peripheral tissue. For example, C-reactive protein, an acute phase protein, considered as a marker of tissue damage and inflammation, 10 and interleukin-6, a proinflammatory cytokine, have been shown to be overex- pressed in AD brain tissue and increased in AD plasma, when compared with controls. 11–15 Plasma also provides an opportunity to assess vascular changes in disease. Hypothesis-driven research from our laboratory demonstrated that plasma levels of oxidized low-density lipoprotein were increased in AD, 16 and other research has reported fibrinogen in plasma to be specifically oxidized in AD. 17 These 2 vascular pathologies are strongly associated with development of atherosclerosis 18,19 and support the notion that atherosclerosis is a risk factor for this dementia type. 20 They also highlight how peri- pheral biochemical changes can play an important role in understanding the pathologies and mechanisms that may underlie AD. Proteomic work using classical 2-dimensional gel electrophoresis (2-DE) has successfully identified altered plasma protein expression patterns in AD, compared with control. In a small cohort, Liu et al 21 showed that serum levels of apolipoprotein A-1 were reduced in AD. The researchers propose apolipoprotein A-1 as a biomarker of AD and suggest that its importance in disease process may involve a reduction in cholesterol levels and Ab formation in AD brain. In a separate study, Hye et al 9 reported a-2 macroglobulin and complement factor H to be increased in AD patients compared with controls. In a later follow-up study by the same group, these 2 proteins were reported to strongly correlate to a hippocampus metabolite measure associated with cognitive decline in dementia. 22 The researchers suggested that these plasma proteins may reflect disease progression in AD. 22 More recently, proteomic study, the plasma proteins serpin F1 and complement 1 inhibitor were reported to be downregulated in mild-to- moderate AD, which indicates 2 more potential candidate biomarkers for this disease. 23 A new avenue of proteomics has recently emerged that couples isobaric tagging with mass spectrometry [eg, isobaric labeling for relative and absolute quantification Received for publication November 29, 2010; accepted July 17, 2011. From the *School of Sport and Exercise Sciences; wSchool of Dentistry; zSchool of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK; and yInstitute of Gerontology and Geriatrics, University Hospital, Perugia, Italy. The Thermo Scientific LTQ Orbitrap Velos ETD used in this research was obtained from the Birmingham Science City Translational Medicine: Experimental Medicine Network of Excellence project, with support from Advantage West Midlands (AWM). The authors declare no conflicts of interest. Reprints: Sarah Aldred, PhD, School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK (e-mail: s.aldred.1@bham.ac.uk). Copyright r 2012 by Lippincott Williams & Wilkins ORIGINAL ARTICLE Alzheimer Dis Assoc Disord  Volume 26, Number 4, October–December 2012 www.alzheimerjournal.com | 329