Borg, Jacques

Campos, Alexandre R.

Diema, Claudio

Guinovart, Joan

Oliveira, Eliandre de

Omeñaca, Nuria

Vilaseca, Marta

Repositorio Institucional de la Universidad de Córdoba

3527(Ï0,&$1Ò0(52)(%5(52 43Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease. ALS is a progressive, fatal, neurodegenerative disease caused by the degeneration of motor neurons, the nerve cells in the central ner-vous system that control voluntary muscle movement. Diagnosis is achieved with clinical evaluation, as well as electromyography and neuroimaging. However, a VLJQL¿FDQWQXPEHURISDWLHQWV DUHPLVGLDJQRVHGRUdiagnosed after a long delay. Therefore, it is crucial WR¿QGSRWHQWLDOELRPDUNHUV WR LPSURYHHDUO\ DQGreliable diagnosis. Using a Bottom-Up analysis and LVREDULFTXDQWL¿FDWLRQRIFHUHEURVSLQDOÀXLG &6)IURP$/6SDWLHQWVZHUHFHQWO\LGHQWL¿HGDSDQHORIdifferentially expressed proteins (Borg et. al, submit-ted). In the present study we applied Top-Down pro-WHRPLFVWRH[DPLQHPRGL¿FDWLRQVRIWKHVHSURWHLQVLQ&6)VDPSOHV3RVWWUDQVODWLRQDOPRGL¿FDWLRQV370and various isoforms of targeted proteins from control and sporadic ALS (SALS) were characterized. ALS has largely unknown pathogenesis that ty-pically result in death within a few years from diag-nosis [1]. There are currently no effective therapies for ALS. Clinical diagnosis usually takes several months to complete and the long delay between symptom onset and diagnosis, limits the possibilities for effective intervention and clinical trials [2]. The establishment of protein biomarkers for ALS may aid an earlier diagnosis, facilitating the search for effective therapeutic interventions and monitoring GUXJHI¿FDF\GXULQJFOLQLFDOWULDOVOur goal was to identify by Top-Down proteomics PTMs and isoform variants of targeted CSF proteins in ALS patients and controls, with the aim of using them DVDGLDJQRVLVWRRO:HIRFXVHGRQSURWHLQVZKLFKZHUHfound differentially expressed in our previous studies of CSF from ALS patients (Borg et. al, submitted). In order to achieve this goal, the following work-ÀRZZDVDSSOLHG )LJXUH&6)SURWHLQ VDPSOHVwere precipitated with acetonitrile [3] and submit-Top-Down proteomic analysis of CSF proteins from ALS patients Claudio Diema1, Alex Campos2, Núria Omeñaca1, Eliandre de Oliveira2, Joan Guinovart3, Jacques Borg4, Marta Vilaseca11Mass Spectrometry Core Facility, 3 Metabolic engineering and diabetes therapy, Institute for Research in Biomedicine, Barcelona, Spain. 23URWHRPLF3ODWIRUP6FLHQWL¿F7HFKQLFDO6HUYLFHV%DUFHORQD6FLHQFH3DUNSpain. 4Medical Faculty Jacques Lisfranc, Jean Monnet University, Saint-Étienne, Franceted to liquid chromatography- mass spectrometry (LC-MS) analysis. In detail, Top-Down analysis was performed by online LC-nanoESI-MS coupling on a /74)78OWUD7KHUPR6FLHQWL¿FZLWKVLPXOWDQHRXVfraction collection using the Triversa Nanomate (Ad-YLRQ%LR6FLHQFHV$%LR6XLWHS3KHQ\O:DWHUV10 µm RPC 2.0 x 75 mm column with a 5-80% ACN gradient over 60 min (100 µl/min) was used for intact protein separations. A 1/250 part from the column eluent was injected directly to the mass spectrome-ter through the Triversa Nanomate using nanoESI chip technology (on line LC-nanoESI-MS, dynamic Top-Down) and the rest of the eluent was fraction collected in a multi-well plate through the same Na-nomate.  According to data obtained by dynamic 7RS'RZQWKHVSHFL¿FUHWHQWLRQWLPHVRIP]LRQVRILQWHUHVWZHUHYLVXDOL]HGDIWHUZDUGVWKHVHVSHFL¿Ffractions containing ions of interest were infused into the MS from the multi-well plate and analyzed by static Top-Down. Protein fragments were obtained by applying CID or ECD and/or IRMPD on selected intact proteins ions according to our interest of iden-WL¿FDWLRQ370VWXGLHV From 500 to 1000 microscan averaging was necessary to increase signal to noise ratio (S/N) and obtain informative fragment spectra from infused fractions. Obtained data was processed E\3URVLJKW3&7KHUPR6FLHQWL¿FIn this work, we focused mainly in 4 proteins na-med apolipoprotein A1, cystatin C, ȕ-microglobulin and ȕ-microglobulin short isoform.A truncated isoform of apolipoprotein A1 (Apo A1) was observed in both control and SALS sam-ples. An isoform of -128 a.m.u of this truncated Apo A1 form was also detected in both samples and fragment analysis was consistent with K-10 deletion (natural variant; K missing in Marburg/Munster 2 isoform). Short and major isoforms of ȕPLFURJOREXOLQZHUH HQFRXQWHUHG6HYHUDO IUDJ-PHQWV LQGLFDWHG DPRGL¿FDWLRQ RI UHVLGXH DW SR-44 3527(Ï0,&$1Ò0(52)(%5(52sition 22 (pyroglutamic acid formed from Gln), a glycosylation at Ser4 or Ser2 and a phosphorylation in the short isoform. Moreover, further analysis of cystatin C protein showed a truncated isoform, an oxidation of the Met14 and a phosphorylation. Table 1 summarized PTMs characterized by Top-Down. Protein samples collected in multi-well plate at re-tention times of interest were pooled and digested ZLWKVSHFL¿FHQ]\PH7KHVHSHSWLGHVZHUHDQDO\]HGDSSO\LQJQDQR/&0606DQGWKHLGHQWL¿HGSURWHLQVFRQ¿UPHGWKHUHVXOWVREWDLQHGE\7RS'RZQFigure 1. :RUNÀRZDSSOLHGIRULGHQWL¿FDWLRQDQGFKDUDFWHUL]DWLRQRISRWHQWLDOELRPDUNHVIRU$/6The isoform variants and PTMs detected in this work, showed the potential of Top-Down applied in ELRPDUNHU¿HOG1HYHUWKHOHVVWKHVHUHVXOWVPXVWEHvalidated in order to use them as a diagnosis tool for ALS using and independent methodology.Acknowledgements: The authors would like to acknowledge Michaela Scigelova and Vlad Zabrous-NRYIURP7KHUPR6FLHQWL¿FIRUWKHLUDVVLVWDQFH:Hthank Mark Baumert and Kees Vlak from Advion Biosciences for technical support and advice.Table 1. Summary of the PTMs characterized by Top-Down methodology.Proteinret.times Intact mass**Major isoform detectedcontrol ALSPTMsApo A1ß-microglobulint, -128.09, missing K107 or term Qtruncated Apo A1(t)t, +162.9, glycation-17.02, pyro-Glu from Q-17, +79.96 phosphorylation-17, +162.05, Glc at S2/4truncated cystatin C (t)t, +15.99, M(O)t, +79.96, phosphorylation31.2-31.724.8-25.2 13705.9111741.02ß-microglobulin(short isoform)cystatin C21.2-21.923.5-24.530758.9328061.4727933.4328061.4028224.4428384.4813334.4813350.5613413.5113334.4813350.5613413.5111721.7811801.7611883.8411721.7811801.7613752.8313871.8213928.8413871.8413928.8615789.0813334.5627933.4328061.4028386.633527(Ï0,&$1Ò0(52)(%5(52 45References [1] Rowland L.P., Shneider N.A. Amyotrophic late-ral sclerosis. N. Engl. J. Med, 2001; 344:1688–1700.[2] Ryberg H and Bowser R. Protein biomarkers for amyotrophic lateral sclerosis. Expert Review of Proteomics, 2008: 5: 249-262.[3] Abdi F, Quinn JF, Jankovic J et al. Detection of biomarkers with a multiplex quantitative pro-WHRPLFSODWIRUPLQFHUHEURVSLQDOÀXLGRISDWLHQWVwith neurodegenerative disorders. J Alzheimers Dis. 2006;  9:293-348. Introduction Tissue proteomic studies on atherosclerosis have traditionally focused on whole artery extracts from biopsy or necropsy origin. Arterial intima and media layers are both involved in atherosclerotic development. In the present work, we describe an optimum method which employs the combination of Laser Microdissection and Pressure Catapul-ting (LMPC) and 2D-DIGE saturation labelling to investigate the human intima and media sub-proteomes isolated from atherosclerotic (coronary and aorta) or non-atherosclerotic (preatherosclero-tic coronary) arteries.MethodsCoronary biopsies from patients undergoing bypass surgery and coronary and aorta necropsies were immediately washed in saline and freezed em-bedded with OCT. Intima and media were isolated by LMPC with a Microbeam System (PALM Micro-laser). Several methods for staining (hematoxilin, cresyl violet), protein extraction and DIGE labelling were tested in order to achieve the better protocol Optimum method designed for 2D-DIGE of arterial intima and media isolated by laser microdissectionFernando de la Cuesta1, Gloria Álvarez-Llamas1, Irene Zubiri1,  Aroa Sanz-Maroto1, Alicia Donado2,Luis Rodríguez-Padial4, Ángel García-Pinto2, María González-Barderas*5, Fernando Vivanco1,61Department of Immunology. Fundación Jiménez Díaz, Madrid, Spain. 2Cardiac Surgery Unit. Hospital Gregorio Marañon, Madrid, Spain. 3Department of Pathology. Hospital Virgen de la Salud, Toledo, Spain. 4Department of Cardiology. Hospital Virgen de la Salud, Toledo, Spain. 5Department of Vascular Physio-pathology. Hospital Nacional de Paraplejicos, SESCAM, Toledo, Spain. 6Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain.for 2-DE analysis of human arterial layers. First of all, a spin column chromatographic step with Pro-tein Desalting Spin Columns (Pierce) was assayed in order to minimize negative effects from remai-ning stain. In addition, different lysis buffers were WHVWHGWRDVVXUHHI¿FLHQWH[WUDFWLRQRIDUWHULDOOD\HUVproteome, which is problematic due to highly acidic P\R¿ODPHQWSURWHLQVDQGSUHVHQFHRIFDOFLXPResultsConcerning staining methods, both hematoxilin and cresyl violet were found to negatively affect protein extraction and IEF. A chromatographic spin column step prior to saturation DIGE labelling im-proved substantially 2-DE gels, permitting the use of both stains for human arterial layers proteomic analysis. Ethanol solved cresyl violet minimizes proteases action and facilitates visualization of arte-rial tissue. For this reasons this stain was chosen for further analysis. Optimal DIGE labelling conditions were set at 1nmol TCEP and 2nmol Dye, so that free dye deleterious effects were minimized. The addi-tion of SDS, and in a higher extend DTT, on lysis EXIIHUVLJQL¿FDQWO\LQFUHDVHGSURWHLQVROXELOL]DWLRQ

Top-Down proteomic analysis of CSF proteins from ALS patients

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