METHODOLOGIES WORKSHOP

Tuesday, September 30, 2008 1-5 pm

A new feature of the 2006 gathering, the methods workshop, will be offered again on Tuesday afternoon, September 30, for researchers interested in the state of the art. It includes introductory and advanced tutorials and hands-on proteomics science. The OPTM 2008 Methods Workshop curriculum will cover the complex of mass spectral technologies that the Core Proteomics Laboratory is fashioning into one overall proteomics approach, two-thirds lecture and one-third wet lab. The workshop will be held in conference rooms adjacent to the auditorium; attendance is limited to 45 in order to ensure the appropriate environment. The technologies that will be covered include:

Electrospray Fourier Transform (FT) Mass Spectrometry (MS) to provide the mass of intact protein samples as well as top-down sequencing and initial peptide mapping;

Matrix-Assisted Laser Desorption/Ionization Time-of- Flight MS for peptide mass mapping;

MALDI-FT MS for more accurate peptide mass measurement;

Laboratory detection of Thiol modifications, iodoacetamide labeling, synthesis of biotin-tagged glutathione and other labels, S-Glutathiolation, and antibodies for oxidized thiols;

Proteome Lab Protein Fraction 2-Dimension (PF2D) protein fractionation methods to analyze protein abundances and detect protein modifications;

Capillary Liquid Chromatography MS and MS/MS to automate sequencing, variant identification, and identification of post-translational modifications;

Use of MALDI and ESI hybrid FTMS instruments constructed in-house within Boston University School of Medicine’s Mass Spectrometry Resource, an NIH-NCRR Research Resource. The present BUSM instrumentation and the new cryoFTMS now under construction at the CPC will be described in the tutorial, as well as the use of commercial instruments.

Lunch will be served for workshop participants at 12:15 pm, prior to start of the session.

Wednesday, October 1, 2008

8:00 am            Registration (inner lobby of Auditorium)

Session 1:         THIOL OXIDATIONS I, Richard A. Cohen and Aruni Bhatnagar, chairs (Auditorium)

8:30      Keynote presentation, “Thiol Reactivity and the Identification of Cellular Oxidant Targets,” Christine C. Winterbourn, Ph.D., Professor of Pathology, University of Otago, Christchurch, New Zealand.

9:10       Jiang Y, Budnik M, Théberge R, Prokaeva T, Bodi K, Seldin D, SkinnerM, Connors LH, Costello CE.  Characterization of Amyloidogenic Immunoglobulin Light Chains, PTMs

9:30      Zang T, Klaene J, Liu S, Chen D, Dhamnaskar K, Zhou ZS. Development of Protein Chemistry Methods for the Global Analysis of Protein Homocysteinylation and Deamidation

9:50      Tong XY, Cohen RA. NOX4 Oxidizes SERCA and Induces Vasculature Dysfunction in a Prediabetic Model

10:10    Hill BG, Landar A, Darley-Usmar VM. Mitochondrial Redox State Regulates Electrophilic Protein Modification

10:30    Break 

10:50    Burgoyne JR, Mayr MM, Eaton P. S-Nitrosylation: A Novel Mechanism for the Activation of PKG1a and Type I PKA

11:10     Evangelista A, Weisbrod RM, Tong XY, Cohen PA. VEGF-In­duced Endothelial Cell Migration is Modulated by Oxidative Modification of the SERCA Cysteine-674 Reactive Thiol.

11:30    Oh, JY, Landar A, Darley-Usmar V. Cellular Responses Medi­ated by the Cyclopentenone 15-Deoxyprostaglandin J2 (15D-PGJ2); A New Model for Signal Transduction by Reactive Lipids

 11:50   Valsky E, Dumlao T, Siwik DA, Cohen RA, Colucci WS.4-Hy­droxynonenal Acutely Activates SERCA in Cardiac Myocytes via S-Glutathyolation of Cysteine-674

 12:10   Discussion

 12:30   Luncheon and Poster Session (Hiebert Lounge)

                Tours of Cardiovascular Proteomics Center and Mass Spectrometry Resource

Session 2:        THIOL OXIDATIONS II, Santiago Lamas and Christine C. Winterbourn, chairs (Auditorium)

2:30      Keynote Presentation, “The Biological Effects of Mitochondrial Protein Thiol Modification by Electrophiles,” Aimee Landar, Ph.D., Assistant Professor University of Alabama-Birmingham.

3:10       Shonsey E, Renfrow M, Barnes S. Substrate Inhibition of Active Site Residue Modification by an Electrophilic Lipid—Structural Consequences to Enzyme Action.

3:30      Maron B, Zhang YY, Handy D, Beuve A, Tang SS, Loscalzo J, Leopold J. Hyperaldosteronism Impairs Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells.

 3:50     Baba S, Kaiserova K, Conklin DJ, Bhatnagar A. Ischemia-In­duced Activation and Oxidation of Aldose Reductase is Modulated by GSTP Dependent Glutathiolation.

 4:10      Zee R, Pimentel DR., Hou X, Zang M, Yao C, O’Connor PB, Bachschmid MM, Cohen RA. Sirtuin-1 Zinc Thiolate Center is a Direct Molecular Target of Oxidants.

4:30      Kumar V, Kitaeff N, Hampton M, Cannell M, Winterbourn C. Cardiac Ischemia Leads to Selective Oxidation of Mitochon­drial Peroxiredoxin 3.

4:50      Lancel S, Qin F, Trucillo MP, Lennon SL, Communal C, Xu S, Weinberg EO, Siwik DA. Dilated Cardiomyopathy and Progres­sive Myocardial Failure in Gαq Mice are Associated with Transcriptional and Ocidative Post-Translational Modifica­tions of RYR2 and SERCA2

5:10       Discussion

5:30      Social hour with poster viewing, refreshments (Hiebert Lounge)

7:00      Conference Dinner at Amrhein’s Restaurant, South Boston

Thursday, October 2, 2008

8:00 am            Registration desk will be staffed (inner lobby of Auditorium)

Session 3:        Protein Turnover and Oxidation, Philip Eaton and Catherine E. Costello, chairs (Auditorium)

8:30      Keynote presentation, “The Assembly of Cardiac Proteosome Complexes: Pick a Partner and Let’s Dance,” Peipei Ping, Ph.D. FAHA, Professor of Physiology and Medicine/Cardiology, University of California/Los Angeles

9:10       Wang Y, Infusini G, Meriin A, Sherman M, Costello CE. Characterization of Interacting Proteins and Protein Post-Translational Modifications from Polyglutamine Complexes by Mass Spectrometry

9:30      Bernevic B, Petre A, Przybylski M. Proteome Analysis for Identification of Degradation and Oxidation in Pig Muscle Proteins

9:50      Perlman DH, Bauer S, Infusini G, Feelisch M, McComb ME, Costello CE. Label-Free LC-MS Quantitation of Nitrite-In­duced Alterations to the Cardiac Proteome Associated with Long Term Preconditioning.

10:10    Drăguşanu M, Brîndusa A, Przybylski M

                Structural Characterisation of Cysteinyl-Nitrosylated Peptides by Affinity-Mass Spectrometry Using a Specific Nitrosocysteine Antibody

10:30    Break

10.50    Stella D, Renfrow MB, Srivastava OP, Barnes S. Sites of Near-UV Induced Singlet Oxygen Oxidation of Human Alpha B-Crystallin.

11:10     Kehasse AY., Perlman DH., Infusini G, Boucher I, . McComb ME., Trinkaus-Randall V, Costello CE. Specific Phosphoryla­tion Sites on Epidermal Growth Factor Receptor (EGFR) Dictate Recruitment of Downstream Signaling Proteins.

11:30    Gao X, Jayaraman S, Gursky O. Mild Oxidation Promotes and Advanced Oxidation Impairs Remodeling of Human High-Density Lipoprotein In Vitro.

11:50    Discussion

12:30-2 pm        Luncheon and Poster Session with Authors (Hiebert Lounge)

Session 4:        Methods for OPTM Analysis, Michael Pryzbylski and Christian Schoneich, chairs (Auditorium)

2:00      McComb M, Perlman D, Ying W, Dauly C, Infusini G, Tong W, Bhatia V,West J, Costello C. Identification of Oxidative Stress Induced Post-Translational Modifications Via an Intact Protein Separation Space 

2:20      Zaia J, Staples GO,Naimy H.  Informatics for Complex Glycan Spectral Datasets

2:40      Margolin Y, Freeman E, Ivanov AR. Towards Efficient Characterization of Dynamic Proteomic Changes Induced by Oxidative Stress

3:00      Lin C, Mathur R, Aizikov K, O’Connor PB Recent Development of a Cryogenic FT Mass Spectrometer.

3:20      Held JM, Danielson SR, Atsriku C, Benz C, Gibson BW.OxMRM: An MRM Technique to Quantify Redox Status of Endogenous Cysteines.

3:40      Martinez M, Ischiropoulos H, Blair I. Site-Specific Identificationof 3-Nitrotryosine in Platelet Proteins.

 4:00     Theberge R, Tong WW, Infusini G,McComb ME, Costello CE. LTQ-Orbitrap MS Top-Down Analysis of Plasma Proteins: A Model Study with Transthyretin.

4:20      Perdivara I, Deterding LJ, Tomer KB, Przybylski M. Mass Spectrometric Identification of Oxidative Modifications of Tryptophan Residues in Monoclonal Antibodies: Artifact or True Post-Translational Event.

 4:40     Tong W, Perlman D, Weng Z, Costello C, McComb M.

                BUPID: Probability-based Protein Identification by Searching Ssequence Databases Using Peptide Mass Fingerprint Data

 5:00     Discussion

 5:30     Social hour with poster viewing, refreshments (Hiebert Lounge)

Friday, October 3, 2008

8:00 am            Registration desk will be staffed (inner lobby of Auditorium)

Session 5:        OTHER OXIDATIONS, Victor Darley-Usmar and Bradford W. Gibson, chairs (Auditorium)

8:30      Schöneich C, Mozziconacci O, Nauser T. The catalytic Role of Thiyl Radicals in Protein Damage.

9:00      Lubos E, Kelly N, Mahoney C, Zhang YY, Leopold J, Loscalzo J, Handy D. Glutatathione Peroxidase-1 Modulates the Effect of Inflammatory Signals in Endothelial Cells.

9:20      Zhang J, Lancel S, Evangelista A, Trucillo MP, Siwik DA, Cohen RA, Colucci WS Nitroxyl Activates SERCA in Cardiac Myocytes via Reversible S-Glutathiolation of Cysteine-674.

 9:40     Loya F, Rao S, Haile A, Bedja D, Gabrielson K, Wittstein I, Champion HC. Effect of Acute Catecholamine Hyperstimula­tion of the Heart: A Model of “Broken Heart Syndrome”

10:00   Petre BA, Tu T, Gross M, Przybylski M. Molecular Recognition Specificity of Anti-3 Nitrotryosine Antibodies Revealed by Affinity-Mass Spectrometry and Immunoanalytical Methods.

10:20    Redondo-Horcajo M, Martinez-Ruiz A, Martinez P, Rial Eduardo, Vazquez J, Lamas S. Cyclosporine A Increases Mitochondrial Superoxide Production and Inactivates MnSOD by Nitration of Tyr34

10:40    Przybylski M, Petre A, Dragusanu M, Ulrich M, Doering G. Identification and Molecular Structure Determination of Protein Nitrations by Affinity-Mass Spectrometry.

11:00    General discussion, conference summary and conclusion

12:00   Session 6: GRAND ROUNDS (Keefer Auditorium)  OPTM symposium participants join Grand Rounds in the School of Medicine, “Oxidative Stress and Cardiovascular Disease: Focus on Cyclophilin A” presented by Bradford C. Berk, M.D., Ph.D., CEO, University of Rochester Medical Center, Senior Vice President for Health Sciences