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MestReNova Manual © 2009 MESTRELAB RESEARCH Last Revision: 29-Oct-2009 MestreNova 6.0.2 by MESTRELAB RESEARCH This is the manual of MestreNova 6.0.2 MestreNova Manual © 2009 MESTRELAB RESEARCH All rights reserved No parts of this work may be reproduced in any form or by any means - graphic, electronic, or mechanical, including photocopying, recording, taping, or information storage and retrieval systems - without the written permission of the publisher Products that are referred to in this document may be either trademarks and/or registered trademarks of the respective owners The publisher and the author make no claim to these trademarks While every precaution has been taken in the preparation of this document, the publisher and the author assume no responsibility for errors or omissions, or for damages resulting from the use of information contained in this document or from the use of programs and source code that may accompany it In no event shall the publisher and the author be liable for any loss of profit or any other commercial damage caused or alleged to have been caused directly or indirectly by this document MestreNova Manual Table of Contents Foreword Part I Introduction System Requirements Windows System Requirements Mac OS X System Requirements 10 Linux System Requirements 10 Part II Mnova 6.0 12 Part III Installation Guide 15 Part IV Shortcuts 22 Part V Fast Visual Guide to process routine 1D-NMR experimental data 26 Part VI Fast Visual Guide to process routine 2D-NMR experimental data 35 Part VII The Mnova interface 40 Multipage Software 41 Working on a Single Page 44 Mnova Menus & Toolbars 46 Intelligent NMR Clipboard 66 Properties 69 Preferences 82 Contextual Menus 86 Toolbars Customization 93 Localization 99 10 Layout Templates 101 11 How to measure coupling constants? The Crosshair Tool 107 12 Zooming 109 Full View Create Expansions 114 116 13 Cutting Spectra 117 14 Increasing and Decreasing Intensity 119 15 Undo & Redo 119 16 Graphics and Annotations 120 © 2009 MESTRELAB RESEARCH Contents Part VIII Processing Basics 123 1D NMR Processing Tour 123 2D NMR Processing Tour 128 Stacked NMR Processing 136 Fourier Transform 146 FID Shift Diagonal Suppresion Signal Suppresion Apodization Truncation Zero Filling and Linear Prediction Drift Correction The FT Command 149 150 150 151 161 161 164 165 Phase Correction with Mnova 166 Baseline Correction with Mnova 173 Symmetrization 181 Tilt 45 182 Reducing t1 Noise 183 10 Covariance NMR 183 11 Normalization 193 12 Binning 194 13 Align 195 14 Compression 196 15 Smoothing 196 16 Resolution Booster 200 17 Inverting, Transposing and Reversing Spectra 203 18 Arithmetic 203 19 Automated Processing 210 Part IX Analysis Tools 214 Chemical Shift Referencing 214 Peak Picking 218 Peak Picking Properties 234 Integration 235 Integral Properties 250 Multiplets analysis 252 Multiplets Properties 278 GSD (Global Spectral Deconvolution) 281 Line Fitting (Deconvolution) 292 Line Fitting Properties 302 J-Correlator 303 Bayesian DOSY Transform 305 © 2009 MESTRELAB RESEARCH MestreNova Manual Data Analysis 314 10 Molecular Structure 324 Molecular Structure Assignment 328 11 NMR Prediction 332 Modgraph NMR Predict Desktop NMRPredict Server-Based Predict & Highlight Predict & Compare Predict & Verify 12 Tables 333 344 348 351 352 359 13 Spin Sim 372 Part X Scripts 378 Connecting scripts with Mnova objects 385 Connecting scripts with Mnova GUI 394 Mnova script reference 394 Mnova Properties 463 GUI Actions 468 Running Scripts from the command line 473 Scripts Samples 474 Part XI Tutorials 501 Importing Spectrum Parameters 501 How to carry out assignments? 513 Manual Phase Correction in 2D-NMR 520 Show Traces in 2D-NMR 524 Basics on Arrayed-NMR Data Analysis 530 GSD: Applications on DOSY processing and Peak Alignment 547 Superimposed Spectra 554 Pasting Spectra into Reports 556 Mnova for metabonomics 561 10 How to predict a spectrum with NMRPredict Desktop? 569 Part XII Mass Plugin (Windows Only) 576 First Steps with the Mass Plugin 578 Working with the MS plugin 587 Mass Analysis Spectrum Selection Mode Elemental Composition Molecular Match Properties 599 599 605 610 614 © 2009 MESTRELAB RESEARCH Contents Index © 2009 MESTRELAB RESEARCH Part I Introduction Introduction Welcome to MestReNova (Mnova) Mnova is the latest Nuclear Magnetic Resonance and LC/GC/MS data processing, visualization, simulation, prediction, presentation and analysis software package available on the market This Help Manual introduces you to this new software, ensuring that you can use it successfully and achieve good results right from the start We strongly suggest that you print this manual and follow it when first using Mnova, as this will make your introduction to the software very simple and painless, and will make sure that you quickly identify all the main functionality in the software Mnova is the natural evolution of our very popular application MestReC However, it is more than a new version of MestReC Whilst including all the NMR processing and analysis functionality present in MestReC, it is a completely new development and a completely new concept which will open up a whole new range of possibilities when storing and sharing data within organizations, changing the way in which scientists process and report NMR and LC/GC/MS data Mnova has been designed to make NMR and LC/GC/MS both more convenient and more powerful, and to give the user improved results with minimum effort The program provides a variety of conversion facilities for most NMR and LC/GC/MS spectrometer and chromatogram formats and includes all the conventional processing, displaying, and plotting capabilities of an NMR program, as well as more advanced processing techniques with a very easy-to-use and intuitive graphical interface To read a little bit more on the main features, and familiarize yourself with the principles behind the program, follow this guide 1.1 System Requirements Before installation, please read this chapter In this section you will find information on which hardware and software requirements must be met before you can install Mnova In any case, it is worth noting that Mnova is a multiplatform software which will run alternatively on Windows, Mac OS X, Linux and Unix-like systems From now on, multiplatform laboratories will be able to allow each researcher to work on their Operating System of choice, and to seamlessly share data and processing capabilities with other researchers working on different platforms There is no longer a need to have three different software packages, and three different file formats, for different Operating Systems The minimum and recommended system requirements for the software are: Windows Mac OS X Linux 1.1.1 Windows System Requirements The minimum recommended configuration for Mnova installation is at least Pentium 300 MHz, 128Mb RAM, a VGA color monitor with 800 x 600 pixels resolution, a compatible mouse and Windows XP as operating system However, ideal system requirements for optimum operation of the software are Pentium 1,6 GHz or higher CPU with 512 Mb or more of RAM memory and Windows XP, Server 2003 or Vista Mnova will not install under Windows 98 or lower The default installation requires about 75 Mb of disk space If you are going to install (or uninstall) the program on a Windows XP system, be sure you have administrator privileges, because the installation procedure is going to install several files in your © 2009 MESTRELAB RESEARCH MestreNova Manual system folders If you don't have such privileges, you won't be able to install or uninstall the program correctly 1.1.2 Mac OS X System Requirements Mnova requires at least Mac OS X 10.4, with PowerPC or Intel architecture The current version of Mnova is also compatible with Mac OS X 10.5 (Leopard) and and 10.6 (Snow Leopard) 1.1.3 Linux System Requirements Mnova requires at least Pentium 300 MHz, 128Mb RAM, Video Adapter Super VGA (800 x 600) with X Library (Xlib) Please, be sure to choose the correct setup file for your Operating System (Debian Etch, OpenSUSE 10.1 and 11, Fedora Core and 10, Ubuntu 6.06-Dapper Drake and 8.04 Hardy Heron, Red Hat Enterprise or and Mandriva Linux 2007 and 2009) 10 © 2009 MESTRELAB RESEARCH MestreNova Manual significantly less than ppm may be required The algorithm that is used exhaustively computes all possible combinations of elemental formulae subject to the constraints provided by the user (see below) In some cases, millions of possible formulae are evaluated, but because of the speed of the algorithm, this happens very quickly This feature will be very useful to calculate the elemental composition of one or more mass peaks of a spectrum, if the m/z values are of high accuracy, typically ppm mass accuracy or better First of all, you should establish the constraints of the calculation, just by clicking on the corresponding option of the menu: Clicking on 'Constraints' will display this dialog box: 606 © 2009 MESTRELAB RESEARCH Mass Plugin (Windows Only) From the above dialog box, it will be possible to add more 'Element Constraints' just by clicking on this icon You can also select the 'Double Bond Equivalence', 'Tolerance', 'Maximum Result Count', 'Electron Mode', 'Charge State', 'Adduct' and Fitness Threshold' Double Bond Equivalence (DBE): Rings + double bonds Tolerance: Tolerance for matching computed composition to the target m/z If spectral mass accuracy is expressed in ppm, then the tolerance for matching should also be in ppm The typical values in Da should be 0.01 or less, or in ppm or less Tolerances much larger than this, generally give too many hits to be useful © 2009 MESTRELAB RESEARCH 607 MestreNova Manual m/z: symbol used to denote the quantity, in units of Daltons (Da), formed by dividing the mass of an ion in unified atomic mass units by its charge number (regardless of sign) Maximum Result Count: The maximum number of results to return Electron Mode: Electron mode can be even, odd, or both Typically, EI (electron impact) produces positively charged, odd electron ions, whereas ESI (electrospray ionization) produces even-electron ions with either a positive or negative charge EI: Ionization of an atom or molecule by electrons that are typically accelerated to energies between 10 and 150 eV in order to remove one or more electrons from the molecule The term electron impact is deprecated ESI: A process in which ionized species in the gas phase are produced from a solution via highly charged fine droplets, by means of spraying the solution from a narrow-bore needle tip at atmospheric pressure in the presence of a high electric field (1,000 to 10,000 V potential) Charge State: is a signed value The magnitude (1, 2, 3, ) is a function of the experimental ionization technique, while the sign (+/-) is determined by the mode in which the experimental spectrum was produced and detected For electron impact (EI) spectra, charge state is usually +1, while for ESI charge state is usually +1 or -1 for most organic compounds Peptides may have higher charge states Adduct: Adducts are typically found in soft ionization techniques such as ESI Proton (H) is most common for organic compounds, while H and/or Na (or other Group 1A alkali metal cation) may be found in protein, peptide or polymer spectra NOTE: If an adduct is specified, the mass (* charge state) is subtracted from the target mass before compositions are computed The composition calculation assumes a neutral (uncharged, non-adducted) mass Fitness Threshold: Results that match the other criteria but have fitness lower than this threshold will be discarded Fitness ranges from 0.0 (no match to experimental spectrum) and 1.0 (perfect match) Once you have finished with the constraints, click on the 'Elemental Composition' icon and select with the crosshair the desired peak of the Mass spectrum A table with the results will be displayed (it is also possible to find this table under the 'View/Tables' menu) All the results obtained will satisfy the composition, DBE, and tolerance constraints, and will be ranked in decreasing order of fitness Please bear in mind that a fitness value of will be the perfect match 608 © 2009 MESTRELAB RESEARCH Mass Plugin (Windows Only) Please bear in mind that mass spectrometers measure monoisotopic m/z values which is different with the Molecular weight value (calculated using the average mass of each element weighted for its natural isotopic abundance) For example, for C20H23N, the chemical mw is 277.402, and monoisotopic mw is 277.182 Here you can see a list of the exact mass of some elements: Element Symbol Exact Mass Hydrogen H 2H (D) 12C 13C 14N 15N 16O 17O 18O F 28Si 29Si 30Si P 1.00783 2.0141 12 13.0034 14.0031 15.0001 15.9949 16.9991 17.9992 18.9984 27.9769 28.9765 29.9738 30.9738 Carbon Nitrogen Oxygen Fluorine Silicon Phosphorus © 2009 MESTRELAB RESEARCH 609 MestreNova Manual Sulphur Chlorine Bromine Iodine 32S 33S 34S 35Cl 37Cl 79Br 81Br I 31.9721 32.9715 33.9679 34.9689 36.9659 78.9183 80.9163 126.9045 12.2.1.3 Molecular Match Molecule match analysis determines the possible presence of a given structure or structures within a mass spectral data set The molecular ion cluster of each structure is computed and compared to each spectrum in the data set If one or more spectra contain the computed isotope cluster within reasonable matching constraints, the most intense spectrum within the most closely matched chromatographic peak is returned as a positive result Structures not matched are assigned a “not found” result The constraints dialog allows the user to select one or more adducts, specify ion polarity, and other parameters The algorithm makes use of fast isotope cluster calculations and a sophisticated spectral matching technique Molecule match analysis is very general purpose Applications include small molecule confirmation and metabolite identification Limitations are few but include the requirement that the acquired spectra of the compounds being analyzed exhibit at least partial molecular ion clusters The 'Molecule Match' is very useful to see if a mass spectrum and a GC chromatogram match with one of the molecular structures of the document Just load your spectrum (TIC and MS) and paste the molecular structures into any page of the same document (or load whole spectral libraries by opening an sdf file) Next click on the 'Calculate Molecule Match' button of the toolbar 610 (or follow the menu 'Mass Analysis/Molecule Match/Calculate') © 2009 MESTRELAB RESEARCH Mass Plugin (Windows Only) The 'Molecule Match' table with the results will be displayed (it will be also possible to find this table under the 'View/Tables' menu) This table will contain information about the 'Retention Time' (RT), Scan (number of spectrum), Match Score, Similarity, MS Purity, etc Similarity is the comparison of the computed isotope cluster spectrum of a given molecule's molecular formula, plus optional adduct - that is, the mass spectral peaks of the molecular ion - with the mass peaks of the acquired spectrum in that mass region The similarity value can be from to 1, and you will note that a threshold of 0.8 is used For instance, a pesticide named Pydrin, with a molecular formula of C25H22NO3Cl, plus an adduct of H, has an isotope cluster of 420.137 - 100%, 421 140 - 28%, 422.135 - 36%, 423.137 - 10%, 424.140 1.5% This is compared with the mass peaks of the acquired spectrum in the mass range of about 420 to 425 Match Score is the produce of Similarity and MICC value (see below) MICC is molecular isotope-cluster chromatogram Which is the sum of mass chromatograms of all the significant peaks in the isotope cluster Using the example above, m/z 420.137 + 421.140, and so on This is almost the same as the mass chromatogram, across several Daltons, in the case listed above, from m/z 420 425 The reason it is not the same is that for accurately measured mass spectral data, a small tolerance on the order of a few ppm might be used for matching For any given molecule, the MICC chromatogram is normalized to 1.0, and this value is what is used for Match score above © 2009 MESTRELAB RESEARCH 611 MestreNova Manual MS Purity is the proportion of the total intensity of the mass peaks matched in the Similarity measurement relative to the total intensity of the whole spectrum MS Purity is then, the fraction of the found molecular ion intensity versus the total intensity of the spectrum This assumes that most of the intensity of the whole spectrum will be due to the molecular ion cluster, which is true for soft ionization mass spectra, which is true for most forms of ionization in use today Which is not to be confused with MS/MS experiments, which have impart additional energy to produce fragmentation So, using the MS purity as a metric generally provides a useful value that is a larger value with "pure" spectra, that is spectra without a lot of background (or overridden by the intensity of the molecular ion) or without a coelutant Clicking on any "matched molecule" will display the MICC (Molecular Isotope Cluster Chromatogram), overlaid with the TIC The MICC shows the summed response of the isotope cluster for each MS spectrum over the entire data set The MICC is the sum of the mass chromatograms of each m/z value of an isotope cluster In this example, the best result was for the compound number (C20H23N) You can see in the picture below the MICC (in green) over the TIC (in red): In addition, Mnova shows you in the chromatogram the retention time of the match (with a blue vertical line) and displays the corresponding MS spectrum (the number 467 in this case) overlaid with the theoretical one (in green at a M/Z around 278, in this case) It is possible to show/hide the MICC and the theoretic MS just by clicking on the 'View Molecular Match' icon It will be possible to set the 'Molecular Match' settings just by selecting 'Settings' on the 'Mass Analysis/ Molecule Match' menu (or under the Molecule Match scroll down menu): 612 © 2009 MESTRELAB RESEARCH Mass Plugin (Windows Only) This will display the 'Molecule Match Settings' dialog box: Tolerance: To set the m/z tolerance for matching predicted spectra to experimental; by default, MS spectra are matched to either ppm or 0.5 Da, and MS/MS to 10 ppm or 1.0 Da, depending on whether the spectra are derived from high or low resolution instruments Thresholds: Set match score threshold for acceptable result; in most cases the default value of 0.5 is more than enough, however for weak / noisy spectra, the threshold may be lowered Matches per molecule: To set the maximum number of results per molecule; by default, a molecule is allowed to match only once, in which case the best result is returned However, if no MS/MS spectra are available and the sample is known to contain isomers, then increasing this limit will allow the molecule to match every isomer peak Matches per spectrum: Set maximum number of molecules allowed to match a spectrum; by default, each spectrum (or set of related spectra) is allowed to match only one molecule However, if isomers are present in the molecule list, then allowing all matches may be desired © 2009 MESTRELAB RESEARCH 613 MestreNova Manual Adducts: Set adduct/loss pairs using proper atomic or chemical formulas with charge notation Charges: Set maximum charge states to consider By default, +/- are the maxima Dimers: Set to enable consideration of dimers when matching; by default, dimer matching is disabled 12.2.2 Properties Mnova will allow the user to customize nearly all the attributes of the spectrum The user can select the properties of the spectrum by following the menu 'Edit/Properties' or by double clicking the left mouse button or pressing the right mouse button on the spectrum display and selecting 'Properties' from the pop-up menu Double clicking on the spectral window will display the properties dialog box From here you can modify the Line, Title, Peaks, Highlight, Axes and Basic properties of the TIC and MS spectrum by selecting the corresponding tab in the dialog box 614 © 2009 MESTRELAB RESEARCH Mass Plugin (Windows Only) The majority of these properties are similar to NMR spectral properties The most important difference will be in the 'Peaks' tab You can see in the picture below the 'Peaks properties' of the TIC From here, you can 'show the wedges' and the 'baseline' (and select the threshold) or add a second line in the peak label, with information about the Intensity, the Height from the baseline or the Area Please bear in mind that the Area can not be used as a quantitative value © 2009 MESTRELAB RESEARCH 615 MestreNova Manual You can see below the 'Peaks Properties' dialog box of the MS spectrum In this window, you can change the Color, Font and Number of Decimals of the Peak label and you can also show/hide the 'Second Line Label (which can contain information about the 'Intensity' or the 'Relative Abundance') 616 © 2009 MESTRELAB RESEARCH Mass Plugin (Windows Only) Selecting Title in the tabbed menu, will display the below window: © 2009 MESTRELAB RESEARCH 617 MestreNova Manual Clicking on this icon 'RT' or 'Scan ranges': 618 will allow you to customize the format of the title; for example to display the © 2009 MESTRELAB RESEARCH Mass Plugin (Windows Only) Here you can see an example of a TIC and MS after having changed some properties: You can read more about general Mnova properties here © 2009 MESTRELAB RESEARCH 619 Thank you! ‘Thank you for reading this Manual, and for purchasing this release version of MNova We will be very keen to read your feedback on the application, to hear about any bugs you may find and to also listen to any additional ideas or suggestions you may have Please remember that you can send all those, and any queries about the software, or requests for help, to: support@mestrec.com Keep checking our web site (www.mestrelab.com) for additional information on our range of software packages, and for news on our company.’ .. .MestreNova 6.0.2 by MESTRELAB RESEARCH This is the manual of MestreNova 6.0.2 MestreNova Manual © 2009 MESTRELAB RESEARCH All rights reserved... command line interface: dpkg -r mestrenova Other (*.rpm): type from the command line interface: rpm -e MestReNova 20 © 2009 MESTRELAB RESEARCH Part IV MestreNova Manual Shortcuts Mnova provides... spectrum M Manual zoom H Fit to Height T Text annotation E Create Expansions K Manual Threshold Peak Picking Ctrl+K Activates Peak by Peak I Activates Manual Integration J Activates Manual Multiplet