RAPID COMMUNICATION Optimization protocol for amyloid-b peptides detection in human cerebrospinal fluid using SELDI TOF MS Valentina Albertini 1 , Anna Bruno 2 , Anna Paterlini 1 , Simone Lista 3 , Luisa Benussi 2 , Cristina Cereda 3 , Giuliano Binetti 2 and Roberta Ghidoni 1,2 1 Proteomics Unit, IRCCS ‘‘Centro S. Giovanni di Dio-FBF’’, Brescia, Italy 2 NeuroBioGen Lab-Memory Clinic, IRCCS ‘‘Centro S. Giovanni di Dio-FBF’’, Brescia, Italy 3 Lab. Experimental Neurobiology, IRCCS, Neurological Institute ‘‘C. Mondino’’, Pavia, Italy Received: August 11, 2009 Revised: September 11, 2009 Accepted: October 7, 2009 Purpose: The aim of the present work was to set up an optimized protocol for human cerebrospinal fluid amyloid-b (Ab) profiling. Experimental design: We devised an immunoproteomic assay that employs monoclonal antibodies (mAbs) on Preactivated Surface (PS20) chip array followed by SELDI TOF MS. A comparison of a number of factors was performed, and the impact of these differences was noted. Each variable was tested using in parallel two different mAbs, 6E10 and 4G8. In addition, we tested whether the combined use of these two mAbs could improve the capture of N and C-terminally truncated Ab peptides and then the quality of spectra. Results: The best results were obtained using a mixture of Ab mAbs (0.125mg/mL 6E1014G8): 15 Ab peptides (including 3 N-terminally truncated forms) were detected. Conclusions and clinical relevance: This approach has many potential advantages in speed, sensitivity and economy of reagents and could be helpful in order to define the role played by specific Ab truncated forms in cognitive decline. Keywords: Alzheimer’s disease / 6E10 and 4G8 / Cerebrospinal fluid / N and C-terminally truncated Amyloid-b peptides / PS20 ProteinChip array Amyloid-b (Ab) plaque deposition is a pathological hallmark of AD, and altered metabolism of Ab and the b-amyloid precursor protein in the brain has been postulated to be the primary cause of AD [1, 2]. The physiopathological proces- sing of b-amyloid precursor protein involved various proteolytic activities leading to a complex set of Ab frag- ments [3, 4]. The predominant protein component of Ab plaques are strongly aggregating peptides with an approx- imate molecular mass of 4 kDa [5, 6]. Among these peptides, Ab1–40 and Ab1–42 have been the dominant focus research, but it is well established that N- and C-terminally truncated or modified forms of Ab peptides also exist in AD brain [7, 8]. Body fluids, such as cerebrospinal fluid (CSF), plasma, serum or urine represent a cellular protein-rich information reservoir that contains traces of what has been encountered during its circulation throughout the body. CSF, which is a continuum of the brain, is an obvious source of markers reflecting central neuropathologic features of the brain diseases. The heterogeneity of brain Ab species was also described in the CSF. The proteolytically processed Ab peptides (other than Ab1–40 and Ab1–42), however, are difficult to detect using standard methods, possibly because they comprise a heterogeneous set of both N- and C-term- inally truncated peptides, some of which are present only at low levels. Some studies used MS for studying human CSF Ab peptides [9–16]. MS allows for detection of a variety of modified and truncated Ab peptides, thus enabling a more detailed and unbiased analysis of fragments that may play a role in neurodegeneration. An immunoproteomic approach – which combines specificity of 6E10 (against Ab epitope Abbreviations: Ab, amyloid-b; AD, Alzheimer’s disease; APP, b-amyloid precursor protein; CSF, cerebrospinal fluid; E, laser energy; FM, focus mass; ON, overnight; RT, room temperature; SPA, sinapinic acid Correspondence: Dr. Roberta Ghidoni, Proteomics Unit, Neuro- BioGen Lab-Memory Clinic, IRCCS ‘‘Centro San Giovanni di Dio- Fatebenefratelli’’, via Pilastroni 4, 25125 Brescia, Italy E-mail: rghidoni@fatebenefratelli.it Fax: 139-030-3533513 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com 352 Proteomics Clin. Appl. 2010, 4, 352–357 DOI 10.1002/prca.200900166