1 Exploring alternative proteases and fragmentation methods for proteomics Albert Heck1 1. Utrecht University, Utrecht, NL, Netherlands Proteome analysis heavily relies on a broad mixture of analytical techniques, starting from sample preparation, to separation and enrichment and last but certainly not in the least mass spectrometry. Through developments in these enabling technologies MS- based proteomics has matured and start to deliver biological relevant information. Still our measurements are still far from comprehensive, and the optimal matured workflow does not (yet) exist for proteomics. In my laboratory we try to explore alternative complementary methods to explore better the richness of the proteome In more detail I will describe methods enabling 1) enhanced peptide coverage based on a combination of HCD and ETD that leads to improved analysis of phosphopeptides, MHC peptides 1 and protein disulfide bridges 2, 2) enhanced protein and proteome coverage, by using multiple proteases and 3) improved analysis of intact proteins and protein complexes enabling to visualize all proteoforms of a given protein at once and following kinase phosphorylation in real-time using top-down approaches 3 1. Mommen et al. PNAS 111 (2013) 4507-4512 2. Liu et al. Mol Cell Prot 13 (2014) 2776-2786 3. Van den Waterbeemd et al. Angew Chemie 53 (2014) 9660-9664 2 Bulking up muscle proteomics: the exercise-regulated phosphoproteome and the control of muscle growth Benjamin Parker2, 1, Nolan Hoffman2, 1, Jonathan Davey3, Rima Chaudhuri2, Kelsey Fisher-Wellman1, Max Kleinert4, Pengyi Yang5, Sean Humphrey6, Daniel Fazakerley2, Paul Gregorevic3, Erik Richter4, David James2 1. Garvan Institute of Medical Research, Darlinghurst, NSW, Australia 2. The University of Sydney, Sydney, NSW, Australia 3. Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia 4. University of Copenhagen, Copenhagen, Denmark 5. National Institutes of Health, North Carolina, USA 6. Max Planck Institute for Biochemistry, Martinsried, Germany Exercise plays an essential physiological role in the regulation of acute and long-term muscle health, insulin sensitivity and whole body metabolic homeostasis. However, only a few signalling pathways and downstream substrates are known to trigger these beneficial effects. We performed the first mass spectrometry (MS)-based phosphoproteomic analysis of human skeletal muscle biopsies obtained from healthy male subjects before and after an acute bout of high-intensity bicycle exercise. We identified 1,004 phosphosites that were significantly regulated with acute exercise. More than 900 of these phosphosites have not previously been associated with exercise and the upstream kinase is unknown. Given the known therapeutic potential of the AMPK pathway, we performed two additional MS screens to specifically pinpoint novel AMPK substrates in the human muscle including: (1) global MS-based phosphoproteomic analysis of myotubes with pharmacological AMPK activation; and (2) a novel global AMPK in vitro kinase assay combined with targeted phosphopeptide quantification with data-independent acquisition MS. Integration of these data sets revealed a number of novel AMPK substrates including A-kinase anchor protein 1 (AKAP1) that was validated in vivo. Phosphosite mutations identified a novel role for AMPK-phosphorylation of AKAP1 in mitochondrial respiration. We next investigated the use of proteomics to identify substrates involved in the regulation of long-term muscle growth. Follistatin, a potent inhibitor of TGFbeta-signalling, regulates muscle hypertrophy through an incompletely understood mechanism. Proteomic analysis of mice muscle-specific and inducible adeno-associated virus (AAV)-mediated overexpression of follistatin was performed over a time-course. Numerous proteins not previously implicated in the regulation of muscle growth were identified including the E3-ubiquitin ligase, ASB2. AAV-mediated overexpression of ASB2 induced significant muscle atrophy highlighting a novel regulator of muscle mass. These data and developed methodology will serve as an invaluable resource for future studies of muscle physiology and the development of novel therapeutics. 3 TWENTY YEARS OF THE PROTEOME Marc Wilkins1 1. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW On Monday the 5th of September in 1994, during the first Siena meeting (2D Electrophoresis – from protein maps to genomes), I presented the concept of the proteome and the term itself. Since that time, the field we now know as proteomics has expanded dramatically. Proteomic technology, and the new experimental paradigms that this technology supports, has revolutionised the way in which proteins can be studied in the challenging context of a cell or tissue. It has provided stunning insights into the diversity of proteins, their expression and many aspects of their function. This presentation will discuss 20 years of the proteome and of proteomics. It will discuss some major achievements of the field, some of the grand challenges that remain, and will speculate a little about what the future might hold. 4 Advent of Proteomics Discourse in Australia: The Lorne Proteomics Symposia Rob Moritz1 1. Institute For Systems Biology, Seattle, WA, United States This year marks the 20th anniversary of the Lorne Proteomics Symposium, an annual meeting of the Australasian Proteomics Society. From its humble beginnings, the APS meeting (for which it is now commonly referred to) has grown into an internationally recognized meeting for proteomics scientists to exchange ideas, start new collaborations, promote new technologies and to gather socially amongst friends and start up new friendships and collaborations on a global scale. The Lorne Proteomics Symposium of the APS is now considered a world-class meeting and has hosted well over 100 international guests over the last 20 years. The concept of the meeting was originally to provide a technology focused day prior to the Lorne Proteins Conference (a world class meeting in itself now in its 40th year. These “technology days” proved to be so successful, the meeting soon found itself expanding to include many aspects of protein identification and quantitation to also promote biological, clinical and agricultural applications of the technology. Lending from the phrase “proteomics” coined by Marc Wilkins for part of its name, this meeting now encompasses all aspects of proteome based studies and has become a fertile and important international meeting as part of the world wide proteome organization. We celebrate the 20th anniversary of the Lorne APS meeting. 5 The Hitchhiker’s guide to Glycoproteomics and the human Immunoglobulome Daniel Kolarich1 1. Max Planck Institute of Colloids and Interfaces, Potsdam, Germany Glycosylation is well known to alter and fine-tune the functions of glycoproteins. A wide range of biological functions has been described such as conferring proteolytic resistance and mediating or inhibiting inter- and intracellular interactions [1]. As a matter of fact, protein glycosylation does not introduce a single, universally definable functionality. Thus detailed knowledge on the protein specific glycosylation and its site distribution is crucial for any systematic investigation of the functional aspects that protein glycosylation is playing on individual glycoproteins. In that context mass spectrometry has become the method of choice for the identification and in depth primary structure characterisation of glycoconjugates. Nevertheless, it lacks the intrinsic ability to easily distinguish structural isomers of isobaric compounds, and the non-template based bio-synthesis of glycans as well as their non-linear structures pose additional challenges in glycan structure assignment by MS. This presentation will give an overview on the peculiarities of protein glycosylation analysis and will focus in particular on advantages, challenges and limitations for in-depth glycoconjugate characterisation by MS. Synthetic glycopeptides, where both the peptide and glycan moieties can be adjusted, have become essentials tools for the systematic investigation, benchmarking and development of novel approaches for qualitative and quantitative glycopeptide analysis [2, 3]. In combination with orthogonal approaches for the analysis of the released N-glycans (porous graphitized carbon nanoLC ESI-MS/MS & CGE-LIF) and O- glycans (PGC nanoLC ESI-MS/MS) [4, 5], a detailed glycomics and glycoproteomics map of the entire human Immunoglobulome (IgA, sIgA, IgD, IgE, IgG and IgM) has been established, revealing the potential type II receptor sites present on human Immunoglobulins. 1. Kolarich, D., Lepenies, B., Seeberger, P. H., Glycomics, glycoproteomics and the immune system. Curr Opin Chem Biol 2012, 16, 214-220. 2. Stavenhagen, K., Hinneburg, H., Thaysen-Andersen, M., Hartmann, L., et al., Quantitative mapping of glycoprotein micro- heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. Journal of mass spectrometry : JMS 2013, 48, 627-639. 3. Kolarich, D., Jensen, P. H., Altmann, F., Packer, N. H., Determination of site-specific glycan heterogeneity on glycoproteins. Nat Protoc 2012, 7, 1285-1298. 4. Jensen, P. H., Karlsson, N. G., Kolarich, D., Packer, N. H., Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc 2012, 7, 1299-1310. 5. Rödig, J., Hennig, R., Schwarzer, J., Reichl, U.,