The Analysis of Solutions and Surfaces using TriVersa NanoMate Mark Allen, Mark Baumert, Reinaldo Almeida Advion, Harlow, UK Outline - How it works - Solution -Infusion - Lipid and Non Covalent Binding -LC coupling and Fraction Collection - Plant Metabolites - Surfaces -Liquid E xt racti on S urf ace A na lys is (LESA) - Liver – G2 ion mobility - Patient Plaques - Lung - Food/Leaves ppgesticide screening -TLC Plate Reader for compound identification TrIVersa NanoMate IMPROVING THE PERFROMANCE OF MASS SPECTROMETRY More time = more sensitivity = more information to identify compounds Over 500 systems WorldWide in Leading Laboratories Multi purpose system IMPROVING THE PERFROMANCE OF MASS SPECTROMETRY LESA – tissue analysis NCNon Cova lent Metabolite Identification Interactions In tact protein Metabolomics Histones Plants Metabolomics Post Translation Modifications DIDrug Impur ities Glycosylation/Glycomics Chinese Medicine Lipids Organic Chemistry Support AtibdAntibody Igg Flexible chip-based ESI system Fast change over IfInfus ion to nanoLC Petroleomics to fraction collection Reaction Monitoring Hydrogen Deuterium Exchange Compatible with most Mass Spectrometers Co-Marketing Agreements Waters Thermo Fischer Bruker AB Sciex and most recently..Agilent Introducing The ESI Chip and TriVersa nanoMate: Array o f 400 in depen den t nanoe lec trospray nozz les The ESI ChipTM – A Stand Alone Source In the ESI Chip, the electric 220 x 106 V/m field around the nozzle tip is formed from the potential V difference between the microfabricated silicon substrate as an integrated counter electrode and the voltage applied to the fluid vithia the con duc tive p ipe tttitte tip. • Incorporation of the ESI counter electrode into the spray nozzle. This is very different from conventional electrospray devices, which define the electric field by the potential difference between the spray device (fluid potential) and the mass spectrometer inlet or atmospheric pressure ionization (API) interface. • As the distance between the electrodes is only a few microns and constttant, an ext remel y st rong and dtblltifildi stable electric field is reproducibly generated, essentially decoupling the ESI process from the inlet of the mass spectrometer. NanoMate Advantages Over Pulled Capillary NanoSpray Conventional pulled capillary NanoMate Advantages • One analysis per capillary • Up to 400 analyses per chip • Manual operation • Fully automated • Great expertise required • Minimal expertise required • Avg. throughput: 8 -10 • Avg. throughput: 100’ s samples/day samples/day • Better data quality and improved • Unpredictable spray stability may quantitation over pulled capillaries compromise data quality • No microscope needed Automated Infusion Samples are Introduced to the Chip Inlet Using Conductive Pipette Tips Silicon chip bearing a 20 x 20 array of 10 μm ID High Voltage nanospray nozzles 850-1500 V Sample MS Orifice 10 μL conductive pipette tip NanoESI flow rates approx 100 to 500 nL/min TriVersa NanoMate Why do our customers use the TVNM ? – Infusion • Long stable spray from small sample volumes • Zero carry-over infusion analyses of simple samples Protein Core Laboratories - protein structure/research Non covalent interactions Intact protein Identification Histone Characterization Post trans lilation mo difii(PTM)Chdifications (PTM) Character iiization Glycosylation Lipidomics (maybe in hospitals and pharma also) Lipid Analysis, Novel High-Throughput Methodology for Lipidome Analysis “High throughput, reproducible and sensitive analysis of total lipid extracts much easier.” Reference: Christer Ejsing PhD Thesis 2007, Molecular Characterization of the Lipidome by Mass Laboratory: Spectrometry, Technische Universität Dresden http://deposit. d-nb. de/cgi- Dr. Andrej Shevchenko bin/dokserv?idn=983638306&dok_var=d1&dok_ext=pdf&filename=983638306.pdf MPI Mol Cell Biol & Genetics Dresden Germany Automated Shotgun Lipidomics – yeast cells “We achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes.” “This analysis provided 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity The lipid metabolic network of S. cerevisiae. Enzymes are annotated by gene name compared with previous (essential genes are indicated in red). The lipid metabolic network was compiled using the Saccharomyces Genome Database (www.yeastgenome.org) approaches.” and references therein. Lipids monitored by absolute quantification are indicated by green circles. Lipids that were only identified are shown by gray circles. Reference: Laboratory: Ejsing CS, Sampaio JL, Surendranath V, Duchoslav E, Dr. Andrej Shevchenko Ekroos K, Klemm RW, Simons, Shevchenko A (2009): MPI Mol Cell Biol & Genetics Global analysis of the yeast lipidome by quantitative shotgun Dresden mass spectrometry. PNAS 106 (7), 2136-2141. Germany Non-covalent Interaction, Compound Screening Determination of the dissociation constants (Kd) for the complexes of two different “… high sample throughput.” proteins with their ligands. “… improving analysis reproducibility.” “… eliminates carryover” “… very small sample consumption” “great potential for rapid screening of compound libraries in drug discovery programs.” Reference: Laboratory: Zhang S, Van Pelt CK & Wilson DB (2003): Quantitative Determination of Noncovalent Prof. David B. Wilson Binding Interactions Using Automated Nanoelectrospray Mass Spectrometry. Anal. Chem. 75, Molecular Biology and Genetics 3010-3018. Cornell University, New York USA Non Covalent Interactions Receptor-Ligand-Interactions Pheromone Binding Protein (PBP) Ligand-bound PBP Bombykol Unbound PBP ght: MPI Biophysical ght: MPI ibpc.mpg.de/ ii Bombyx mori Pictures Copyr Pictures Chemistry, http://www.mp Principle of the Assay: Ratio of ligand-bound PBP / unbound PBP at increasing ESI-MS cone voltages Reference: Laboratory: Hooper AM, Dufour S, He X, Muck A, Zhou J-J, Almeida R, Field LM, Svatos A, Dr. Aleš Svatoš Pickett JA (2009): High-throughput ESI-MS analysis of binding between the Bombyx Max Planck Institute mori pheromone-bindinggp protein BmorPBP1, its p heromone comp onents and some for Chemical Ecology analogues. Chem. Commun. 5725–5727. Jena NanoLC coupling 0.2 - 0.8 nanoLC or µL/min Nanoaquity butt connected via teflon sheath Move to next nozzle in 3 seconds so no sample loss if sprayer plugs mid run Use your own nanoLC columns TriVersa NanoMate Why Do our customers Use for NanoLC? – NanoLC coupling • The most stable nanoESI for direct coupling to nanoLC. • Automated next nozzle in 3 seconds • Fast change from Infusion to nanoLC Peak Area Comparison RT: 3.20 - 3.40 RT: 3.26 NL: 3 .06E5 AA: 415254 TIC F: + c NSI 100 SRM ms2 653.901 90 [906.399-906.401] MS ICIS 80 ESI Chip 08apr10_003 70 60 dance nn 50 Peak Area ~2x 40 Relative Abu Relative 30 20 greater 10 RT: 3. 26 NL: 8 .40E4 AA: 232397 TIC F: + c NSI 100 SRM ms2 653.901 90 [906.395-906.405] MS ICIS 80 70 Other Nanospray 60 50 40 Source 30 20 10 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38 Time (min) Nano-LC/MS in Routine Bioanalysis: Application to Human NGF Biomarker Studies Presented by: Gary A. Schultz, PhD 18 http://advion.com/events/webinars.php TriVersa Spray Sensing Technology High aqueous, higher spray current ~125nA Re-equilibration of analytical column Low aqueous, lower spray current ~40nA 19 TriVersa NanoMate – Lockmass capability Fraction Collection 200µL/min 0,2 µL/min HPLC 5 - 1000 µL/min 199,8 µL/min Nanoelectrospray Infusion 20s / Fraction Æ 67 µL / Fraction Backpressure Probe Robotic Arm ~ 0.2 psi Conductive Pipette 0 – 3 kV TriVersa NanoMate for Higher Flows 320um columns and above Set-up TriVersa-NanoMate Fraction collection into 384 plates (or 96 plates) Robotic arm for fraction collection and later Infusion 240 µL/min incoming flow rate 300 nL/min to nESI chip and MS Low volume splitting ‘T’ ≤ 0.2 % of the flow 22 The Power of Signal Averaging TriVersa NanoMate Users – Fraction ClltiCollection w ithifith infus ion Pharmaceutical Drug Metabolism/DMPK – development Radioactive metabolites Drug Impurities Chinese Medicine More information from comppplex samples. Academia Metabolomics Plant Metabolomics Biotech Antibody IGG Traditional Chinese Medicine Sample Profiling with UPLC/oaTOF/NanoMate John Shockor, Waters Corporation June, 2009 Triversa ACQUITY UPLC/Xevo Qtof MS with MSE Maximum chromatographic resolution,sensitivity, and speed UPLC Nanomate Exact mass analysis with data-rich information MarkerLynx XS data processing Identify leading markers using extended statistical analysis tool such as PCA/OPL Synapt HDMS Fraction Collection with NanoMate Infuse fraction collected back into MS for MSMS analysis for structural elucidation NanoMate can quickly switch between LC, Re-inject Fraction into UPLC/Qtof MS fraction collection and infusion mode for further separation Lockmass compatible Separate isomers, coeluted peaks so that more detailed info about the sample can be revealed Glycomics, high throughput brain ganglioside (GG) analysis NanoMate robot was coupled to a high-capacity ion trap (HCT) mass spectrometer to create a system merging automatic chip-based electrospray ionization (ESI) infusion, “… improved ionization ultrafast ion detection, and multistage sequencing at superior sensitivity. efficiency” NanoESI chip MS experiments enabled the identification of more than 50 glycoforms exhibiting a high degree of heterogeneity in the ceramide motifs and alterations such as O-acetylation. “… improved signal stability” 25 distinct