Introduction to the Oligo HTCS Systems. Novatia, LLC

Transcription

1 Introduction to the Oligo HTCS Systems Novatia, LLC

2 What is the Oligo HTCS and what can it do? Automated ESI/MS system that can analyze up to 14 oligonucleotide samples/day (>3/day with twin autosampler) Can confirm targeted masses, as well as identify expected impurities and degradants Has routine mass accuracy of +/-.1% with standard ion trap MS Accurate mass capability with Orbitrap MS Can analyze oligos >12 bases in length Standard system can switch from HT to detailed LC/MS mode automatically from within a run sequence Automatically processes data from HT or detailed runs and produces web-based reports Can estimate purity based on MS data

3 Oligo HTCS System Components Thermo Ion Trap Mass Spectrometer LCQ or LTQ series ion traps, very robust, sensitive, and easy-to-use MS systems CTC PAL autosampler Single or Twin injection arms Standard system is fully upgradeable to Twin System HT, 96, 384-well, or vial capability Option of One or Two Dionex Dual Gradient Pumps DGP-36SD or DGP-36RS pumps ProMass Deconvolution Software Automated charge deconvolution and reporting of oligonucleotide mass spectral data Optional ProMass HR for HRAM oligo characterization Xcalibur software control for ease-of-use Analytical methods Optimized for HT and detailed LC/MS analysis using negative ion detection Optimized chemistries (buffers) for thorough desalting of samples

4 ProMass Software Features for Oligo MS Analysis Integrated with Thermo Xcalibur data system ZNova algorithm for artifact-free charge deconvolution Automated deconvolution of spectra in LC/MS data processed from the MS sample list Target mass search capability calculated from oligo sequences, including multiple sequences user-supplied explicit masses Can confirm the presence of common impurities and degradants 3, 5, or internal oligo fragments depurination products side reactions adducts Can estimate the sample purity from the LC/MS or LC/UV/MS data Correction of purity estimates based on co-eluters and known adducts Web-based (HTML), Excel, and XML output New ProMass HR product for automated exact mass analysis up to ~6 bases on the Orbitrap product line

5 Intensity What does an ESI mass spectrum of an oligo look like? 1 4-mer DNA oligo ESI mass spectrum Charge series for mass: m/z

6 Intensity Deconvoluted ESI mass spectrum ProMass Deconvolution of 4-mer DNA Mass, Da

7 New Oligo HTCS Ultimate System A Fully Automated System for HT ESI-MS based Oligo QC CTC PAL Autosampler Thermo Ultimate 3 HPLC Thermo LTQ-XL MS Xcalibur data system with ProMass Deconvolution >14 sample/day max throughput

8 Plumbing Diagram Oligo HTCS Ultimate -high-throughput flow injection/desalting mode Load/Wash Gradient Elute Ultimate 3 To MS Not Used HTS-PAL Autosampler Dionex DGP 36 Dual Gradient Pump PumpLeft PumpRight Divert Valve From UV ValveLeft ValveRight PAL Injector x Oligo Trap Columns 2 Analytical Column Waste Lines Dionex TCC 3 Column Compartment To UV

9 New Oligo HTCS Twin Ultimate System A Fully Automated System for HT ESI-MS based Oligo QC CTC Twin PAL Autosampler Dionex Ultimate 3 HPLC Thermo LTQ-XL MS Xcalibur data system with ProMass Deconvolution >3 sample/day max throughput

10 Plumbing Diagram Oligo HTCS Twin Ultimate -Twin HT Injection Desalting Mode Ultimate 3 2 x Dionex DGP 36 Dual Gradient Pumps PumpLeft PumpRight To UV/MS Twin HTS-PAL Autosampler PumpLeft2 PumpRight Stream Selector Valve ValveLeft PAL2 Injector PAL1 Injector x Oligo Trap Columns Waste Lines Dionex TCC 3 Column Compartment ValveRight

11 Oligo HTCS Automated Process Setup run sequence on LC/MS system Import info from CSV file Data file name / sample ID Oligo sequence or MW info LC/MS run and processing methods Optional ProMass processing parameters Put sample plates in PAL autosampler 96 well or 384 well plates in tray stacks Run Samples ~1 min/sample with on-line desalt method single arm PAL ~25 sec/sample with Twin PAL system Automatically process data with ProMass Occurs automatically after each run (real-time processing) Review results from web browser

12 Intensity x 1^3 Example Oligo HTCS Data Acquisition 12 mer LCQ Fleet 25 pmoles injected C:\Xcalibur\data\Archemix\7267\std12b 7/26/27 4:44:53 PM 2.5 um x 1 ul RT: LC/MS elution profile Total Ion Current.12 NL: 9.19E6 TIC MS std12b sec Time (min) std12b #7-12 RT: AV: 6 SB: NL: 2.97E4 T: ITMS - p ESI Full ms [ ] S m/z ProMass Decon Raw ESI/MS spectrum 12-mer mer Expected Mass: Da Observed Mass: Da Mass, Da

13 ProMass Sample Browser 96-well analysis of 5-13 mer oligos Color codes indicate presence or absence of target masses Color-coded wells are hyperlinked to detailed reports Target mass found as major component Target mass found with other significant components or low spectral quality Target mass found, but not as most intense peak Target mass not found

14 ProMass Sample Browser Web-based hyperlinked reports Sample detailed summary Hyperlinked webbased results allow the user to drill down to the appropriate level of detail! Top-level summary Deconvoluted spectrum Results validation

15 Intensity x 1^3 Intensity x 1^3 Intensity Oligo HTCS Mass Accuracy Deconvoluted mass spectra, 25 pmoles injected, LCQ Fleet mer Expected Mass: 599. Da Observed Mass: 599. Da 599 [] Mass, Da 188 [] 6-mer Expected Mass: Da Observed Mass: 188. Da [-133] Mass, Da 12-mer Expected Mass: Da Observed Mass: Da 3615 [-13] 3628 [] [931] 3645 [17] Mass, Da

16 % Intensity % Intensity MALDI and ESI MS Data of a 12-mer Oligo M = Mass (m/z) ESI ProMass MALDI-ToF Deconvolution MALDI-ToF, M = ESI, M = Mass (m/z) 592.3

17 MALDI and ESI MS Data on Fragile Dual-labeled Probe Black Hole Quencher TM M = % Intensity % Intensity dissociation product MALDI-ToF, ESI ProMass M =73.7 MALDI-ToF Deconvolution ESI, M = Mass (m/z) Mass (m/z)

18 Summary Novatia has developed a truly unique integrated solution the Oligo HTCS allowing totally automated oligo MS analysis from sample in to data out Better resolution and mass accuracy with Oligo HTCS and standard ion trap MS vs. linear MALDI-ToF across entire mass range of oligos analyzed ESI-MS is the technique of choice for analysis of long and/or fragile oligonucleotides Sample throughput of ~14-34 samples/day in HT MW confirmation mode, depending on configuration Detailed LC/MS profiling capability via automatic valve switching System is developed around Thermo Scientific Xcalibur data system allowing for many different MS instrument choices The Oligo HTCS is in use in our own lab for contract oligo analysis (we eat our own cookin )