Poster Session B

B.1 Identification and Quantification Method for UCN2 by LC/MS/MS from Plasma/Serum from Mouse Disease Models Shakey Quazi(1), Wei Zheng(1), Olivier Bezy(1) (1)Pfizer Introduction: The identification of corticotropin-releasing hormone (CRH) has led to the discovery of a growing family of ligands and receptors. CRH receptor 1 (CRHR1) and CRHR2 are mammalian G-protein coupled receptors (GPCRs) with high affinity for CRH and the CRH family of peptides. CRHR1 is predominantly expressed in the brain and plays a vital role in the hypothalamic-pituitary-adrenal (HPA) axis stress responses by secreting adrenal corticotropic hormone (ACTH). CRHR2 is predominantly expressed in the periphery and urocortin 2 (UCN2), a CRHR2-specific ligand, has been involved in the regulation of glucose metabolism. Ucn-2, a 38 aa peptide, shows reasonable homology between rat and human CRH (∼34%), Ucn-1 (43%) and Ucn-3 (37–40%). To measure the accurate concentration of UCN2 present in mouse plasma in our disease and intervention preclinical models, we have developed a novel LC/MS/MS method for UCN2 quantification. Methods: The biomarkers were analyzed by protein precipitation using organic extraction of 30 μL mouse/rat plasma, dried and reconstituted by adding 50 mM ammonium bicarbonate pH 8 and digested with trypsin. The tryptic peptides were then injected using nano-HPLC onto PepMap100 C18 pre-column (5mm × 300μm ID, 5 μm, 100 Å, Dionex) followed by chromatographic separation on a PepMap C18 RSLC nanocolumn (15 cm × 75 μm, 3 μm, 100 Å, Dionex). Mobile phase A and B were 0.1% formic acid in 2% ACN/ water and 0.1% formic acid in 90% ACN/water, respectively. The eluate was introduced into a Quantiva triple quadrupole mass spectrometer with EASY-Spray ion sources for optimum sensitivity. The LC-MS/MS data were analyzed and quantify by Skyline software. Data: In order to gain optimal sensitivity and efficiency as a quantitative biomarker a simple organic extraction was found to be the most advantageous method of sample prep which balances having a simple extraction method (as compared to SPE) but one that gave good overall recovery. Novel Aspect: We have developed sensitive and specific quantification methods by LC/MS/MS for the determination of UCN2 in endogenous levels across species in plasma/serum. B.2 Integrating de novo sequencing with sequence database and spectral library search for in-depth analysis of DIA data with PEAKS Xin Chen(1), Clark Chen(1), Julia Hopkins(1), Paul Shan(1) (1)Bioinformatics Solutions Inc, Waterloo, Canada In recent years, data-independent acquisition (DIA) Mass Spectrometry has become increasing popular due to its parallel nature of acquiring all fragment ions for all precursors within a selected m/z range. Recently, a new software release, PEAKS X+ allows for the searching of DIA data by both spectral library as well as direct database searching. This allows the user to benefit from the speed of a spectral library search when one is available but also the unique feature based detection and de novo algorithms inherent to PEAKS X. First, a library search is performed as the first pass of identification. Any spectra that are not matched to a library entry, are searched against a database, and then are de novo sequenced to determine the peptide sequence. Herein, we benchmark our PEAKS X+ library and database search using a publically available dataset against other DIA search software. Using the library search alone, PEAKS compares very well to the published numbers of identifications generated from Open SWATH, Skyline, Spectronaut, identifying ∼900 more proteins than any other software. Similarly, when only using the direct database search of all spectra, PEAKS outperforms both Spectronaut and DIA-Umpire in terms of identifications observed by about ∼500 protein identifications. Combined, the library and database search resulted in the highest amount of peptide and protein identifications. Taken together, this data suggests that PEAKS X+ will be a useful tool to researchers utilizing DIA Mass Spectrometry, looking to get the most identifications out of their data, regardless of the presence or absence of a spectral library. De novo sequencing provides identification of sequence variants and endogenous peptides. B.3 High-Throughput PEAKS Workflow for Large Scale Quantitative Proteomics using PEAKS Online X Jonathan R. Krieger(1), Julia Hopkins(1), Lei Xin(1), Baozhen Shan(1) (1)Bioinformatics Solutions Inc In the last decade, we have seen unprecedented advancements in the field of proteomics. The recent advancement of instrumentation as well as software has led to an ever increasing number of large-scale quantitative proteomic studies. These projects, either research based or clinical based, often require quantitation of thousands of proteins, either by label free (LFQ) or by using isobaric tags such as TMT. PEAKS Online is a new high-throughput protein sequencing software solution that runs on a shared resource, is flexible to scale, and is fully parallelized with the ability to run on any cluster or multi-cluster CPU machine. Herein we describe the use of PEAKS online in analyzing two published data sets; one employing LFQ, and the other employing TMT. In both cases we demonstrate a drastic decrease in processing and search time using PEAKS online compared to other search software. Furthermore, because PEAKS online is based on the same proven de novo algorithms included in PEAKS Studio, we show an increase in the number of identifications observed in these datasets. In summary, PEAKS online provides an efficient, time-effective solution to search large-scale systems biology level proteomic experiments with unparalleled accuracy. B.4 TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease Charlie Mundorff(1), Daniel Lai(1), Nadine H. Elowe(1), Hasmik Keshishian(1), Alessandro Arduini(1), Bryan T. MacDonald(1), Steven A. Carr(1) (1)Broad Institute of MIT and Harvard Loss of function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. Therefore, catalytic inhibition of ADAMTS7 has been proposed as a therapeutic strategy for treating cardiovascular disease. ADAMTS7 is a large secreted enzyme consisting of a metzincin catalytic domain and over 200 kDa of auxiliary domains with extensive post-translational modifications thought to target the enzyme to the extracellular matrix and hone in on proteolytic targets relevant to vascular function. Several proteins interacting with the carboxy terminal domain of ADAMTS7 have been proposed as catalytic substrates, however no verified substrate cleavage sites have been described. To identify ADAMTS7 extracellular substrates relevant to vascular disease, we have compared the secreted proteome of human umbilical vein endothelial cells (HUVEC) and human carotid artery vascular smooth muscle cells (HCA-SMC) expressing either a control luciferase, full-length mouse ADAMTS7 WT or full-length mouse ADAMTS7 E373Q catalytic mutant. Conditioned media from these cell types were collected, processed and concentrated for proteomic profiling of secretome and as input for TAILS. TAILS (terminal amine isotopic labeling of substrates, Kleifeld Nature Protocols 2011) is a method for identifying and distinguishing protease-generated neo–N termini from mature protein N termini. A TMT10 isobaric labeling strategy was employed to improve confidence of detection and quantification. Comparison of Luciferase/ADAMTS7 WT, Luciferase/ADAMTS7 E373Q and ADAMTS7 WT/ADAMTS7 E373Q results enabled us to identify auto-cleavage sites from the expressed mouse ADAMTS7 proteins and define a substrate consensus cleavage site. N-terminal cleavage sites from novel substrates significantly enriched in ADAMTS7 WT samples represent candidate substrates from relevant cardiovascular cell types and may offer biomarkers for ADAMTS7 catalytic activity. B.5 Role of the Particle for Arrangement of Quaternary structure (PAQosome) in assembly/maturation of human protein complexes and networks Benoit Coulombe(1,2), Philippe Cloutier(1), Maxime Pinard(1), Christian Poitras(1), Marie-Soleil Gauthier(1) (1)Montreal Clinical Research Institute, Montreal, Quebec, Canada H2W 1R7, (2)Department of Biochemistry and Molecular Medicine, Université de Montréal, Quebec, Canada H3C 3J7 Since its discovery a little over a decade ago, the Particle for Assembly of Quaternary structure (PAQosome, formerly RPAP3/R2TP/PFDL complex) has emerged as a critical organizer in the biogenesis of several protein complexes and networks such as protein assemblies involved in transcription, mRNA maturation, translation and nutrient-sensitive signalling pathways. Unsurprisingly, evidence is mounting that this chaperone machine may be involved in tumorigenesis, consistent with a role in regulating proliferation. We will present our most recent results that led to the identification of new client protein complexes and novel post-translational modification (PTM)-based modes of regulation of the PAQosome. Similar to what has recently been reported for axonemal dynein complexes involved in cilium motility, we now identify cytoplasmic dynein complexes that are responsible for cargo transport along microtubules as new clients of the PAQosome. We also identified a phosphorylation-dependent association of the PAQosome subunit RPAP3 with preribosome complexes. Additionally, we will report for the first time the identification of a small ORF-encoded PAQosome subunit, along with a possible role in the regulation of downstream gene, asparagine synthetase (ASNS) whose expression is linked to neurological disorders and response to asparaginase, a chemotherapeutic drug used in the treatment of acute lymphoblastic leukemia (ALL). These results define novel aspects of PAQosome function and regulatio

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