Mass Spectrometry Facility Advanced Analysis Centre University of Guelph Presented by: Dr. Dyanne Brewer Mass Spectrometry Facility Manager May 2014

MS Sample Submission Workflow Data Interpretation Sample Submission Sample Preparation Data Acquisition Training Available: GC-MS derivitization Protein digestion GC-MS LC-MSn Metabolomics software Proteomics software Submission Forms: http://www.uoguelph.ca/~bmsf/

Instrumentation Agilent GC-MS Bruker GC-MS-MS Bruker LC-MSn Solid, liquid, gas, SPME Bruker LC-MSn Agilent LC-HR MS-MS GC- gas chromatography LC- liquid chromatography MS- mass spectrometry

Sample Type: Small Molecule Volatile/semi-volatile GC separation MS confirmation MS-MS quantitation Non-volatile LC Separation MS confirmation/formula MS-MS characterize/quantitate

Small Molecule GC-MS Sample Collection Metabolite Extraction Compound Derivitization Gas Chromatogram NIST match to Glycine N,N-bis(trimethylsilyl) trimethyl ester Dr. Mutch UoG HHNS

Small Molecule Mass Accuracy Best ID Source Formula Species m/z Score Diff (ppm) Score (MFG) TRUE MFG C49 H54 Cl5 N8 O9 P (M+H)+ 1105.2269 94.53 -0.33 Dr. Manderville UoG Chem

Small Molecule LC-MS Dr. Kirakosyan Un. Michigan UV 520 nm Pelargonidin 271 Cyanidin 287 Peonidin 301 Delphinidn 303 Malvidin 331 Petunidin 317 Anthocyanins have been reported to be strong antioxidants, as well as potential anti-inflammatory and anticancer agents [3]. In addition, they act as a vasoprotectors [4] and possibly have anti-obesity effects [5,6]. tart cherry intake reduces blood lipids, plasma and tissue inflammation. -peonidin -hexose -deoxihexose Dr. Kirakosyan Un. Michigan

Small Molecule LC-MS Tissue Extract Dr. Kirakosyan Un. Michigan Heart Bladder kidney Brain Tissue Extract Calibration Curve of Peonidin 3-O-glucoside sample name RT [min] Area concentration_peonidin dihexoside_fg/ul 1%heart_1.54g_(5) 10.2 42548 32.6 1%Bladder_0.75g_(1) 12946 3.2 1%kidney_1.4g_(14) 91933 81.7 1%brain_0.94g-(9) 14337 4.6 It is known that health-enhancing properties of anthocyanins cannot be fully understood without comprehensive analysis of their metabolism and tissue distribution. Our working hypothesis is that intake of tart cherry powder for about two weeks will 1) cause tissue deposition of tart cherry anthocyanins in several organs (heart, kidney, liver, brain, bladder, skeletal muscle, prostate, testes and adipose tissues) and 2) some conjugates (sugar moieties) of anthocyanins may play important roles for tissue distribution. We will therefore, confirm and identify the presence of tart cherry metabolites in targeted tissues of rats and also unravel possible post-adsorptive modifications of anthocyanins. Dr. Kirakosyan Un. Michigan

Metabolomics Workflow Separate and Detect MS GC-MS/LC-MS Compare and Stats MS database search targets Compare to database/standard Target MS/MS MassHunter/MPP software In-house Metlin/NIST library or online ID GC-MS data Pathway?

Sample Type: Large Biomolecule Protein Intact characterization Clean-up LC MS confirmation Identification Solution digest In-gel digest LC Auto MS-MS Database search/de novo sequencing PTM id

Protein MW Protein solution Clean-up LC Deconvoluted data Raw data 27,310.4656 amu Dr. Brown UoG Physics

Protein Identification Protein digest LC-Chromatogram Protein Group Protein ID Accession -10lgP Coverage (%) #Peptides #Unique PTM Avg. Mass Description 1 6181 Colicin_E1_Q372C 283.51 69 60 N 21734 Peptide Fragmentation Protein Identification Dr. Merrill UoG MCB

Advanced Analysis Centre Mass Spectrometry Facility Advanced Analysis Centre University of Guelph Manager: Dr. Dyanne Brewer Research Associate: Dr. Armen Charchoglyan Location: Science Complex Rm 1205 Email: dbrewer@uoguelph.ca Phone: x58649 lab x53793 DB office