m U msI Use Qu er ua Ma nt an 1. nua 2 al
Contents
1. Quantitation R R Recalibration Region name Remove complete Remove normalized CSV files Remove understructures Remove xy dat files RMS Normalization S Savitzky Golay smoothing filter Simple moving first quartile Sorted mass spectrum transform Spectrum smoothing Version AB Index 15 16 15 27 32 15 10 19 19 10 24 24 33 13 12 18 22 20 11 18 36 36 36 36 15 10 10 10 10 Page 38 of 39 Spectrum folder Spectra folder Spectra top folder Standard curve T TIC Normalization Z Zoom in intensity Zoom in mass mslQuant 1 2 User Manual 17 19 15 25 25 Version AB Index Page 39 of 392. Spectrum Name ENE Spectum color T mslQuant 1 2 User Manual Version AB Page 29 of 39 Spectra View How to copy the image To copy the graph image to the clipboard select Copy Image in the popup menu and paste it directly to e g Microsoft PowerPoint How to copy the spectra within a specific m z range To copy the spectra in the window to the clipboard select Copy Spectra in the popup menu and paste it directly to e g Microsoft Excel How to select specific spectra peaks Zoom in and display the peaks of interest A To select the Peak cursor click once on the lt Tab gt key Then click on the left side of the peak and the graph will display information of the centroid m z centroid intensity the peak area and the signal to noise ratio EJ Spectra View Control Colex Medulla Pelvis 392 00 392 50 393 00 393 50 394 00 The information about the selected peaks is automatically copied to the clipboard to be pasted to e g Microsoft Excel How to set peak picking parameters To change parameters for peak picking select the Peak Option in the popup menu Peak Option SNR for Peak Detection Peak Group Detection Set Peak Group Detection Peak Group Within 0 5 Edit the signal to noise ratio for peak selection Edit the mass frame to be able to select a group of peaks mslQuant 1 2 User Manual Version AB Page 30 of 39 Image View Image View Image View is a p
3. Daltonics fleximaging Select also the destination top folder where the ROI folders will be created Spectra Normalization Create Structure Create structrue from Regions XML Regions XML File Slide2d Tio30mini0ug j Destination Top Folder C DATA TestSpectra Create structrue from Regions XML Create Structure Information text Progress Click on the button Create Structure to continue To fill the created folder structures with normalized spectra go back to the tab Match Spectra and continue mslQuant 1 2 User Manual Version AB Page 17 of 39 Quantitation Tool Quantitation Tool Quantitation Tool is a program that e Performs a concentration study and creates a standard curve for quantitation e Performs quantitation of tissue ROI and creates an image xml file with substance concentration for each pixel After normalization and creation of average spectra the next step is to perform standard curve and quantitation of experiment tissue Before running this program run Spectra Normalization to create average spectra Create standard curve A Quantitation Tool Create Standard Curve Spectra Top Folder C DATA MSI_Quantitation_Workshop_msIQuant_Data_300ct ctrl Create Peak List Select m z of a Peak Create Standrad Curve Create Quantitation of Tissue Spectra Folder C DATAWMSI Quantitation_Workshop_msIQuant Data 30Qct Tiss E Top Folder Create Quantitation The first ste
4. Evaluation XML Evaluation XML Evaluation is a program that performs calculation of the generated xml file from the program Quantitation Tool The information and calculation of an xml file are e Number of pixels e Average quantified value e Standard deviation STD of the quantified value e Relative standard deviation RSD of the quantified value e Min value of the quantified value e Max value of the quantified value e Pixel deviation analysis PDA e Pixel ratio calculation PRC Before running this program run Quantitation Tool to create image xml file s Perform xml evaluation XML Evaluation Select Image XML File From Tissue or ROI Image xml file C DATA MSI_Quantitation_Workshop_msIQuant_Data_300Qct Tiss Progress Select an image xml file and click on the button Evaluate to evaluate the file The next dialog box will appear AML Evaluation ExpConc_Low_392 0 1 H P Ga Number of Pixels Average value 7 828608 STD 1 976052 RSD 0 252414 Min 4 416482 Max 11 907694 2 176777 Pixel Ratio Calculation 0 278054 mslQuant 1 2 User Manual Version AB Page 23 of 39 XML Evaluation The information in the dialog is automatically copied to the clipboard to easily past into e g Microsoft Excel Below is an example from Excel when the evaluation is pasted A B hs D E F G H l J 1 ExpConc Cx 281 0 5 H P xml 287 113 7996 65 42072 0 5 4877 0 479 3134 66 25685 0 582224 2 3
5. Hide Spectrum from the popup menu Check the spectrum that is going to be hid and click OK How to edit spectra names colors line widths and spectra order To change order of the spectra click on an item and drag and drop the item to the new position in the list To change the spectra names colors line widths and spectra order select the Edit Spectrum Name Color and Line Width in the popup menu mslQuant 1 2 User Manual Version AB Page 28 of 39 Spectra View Set Spectrum Color and Line Width Double Click to Edit Spectrum or Drag and Drop to Sort Spectra Spectrum Mane Line Width 110714 Slide d O _ROO D06Yr053 1107714 Slide d O _ROO D06Yr0O054 110714 Slide2d 0_ROO0 0067 055 1107714 Slide2d 0_ROO 0067 056 To change the name color and line width of a spectrum double click on the item and the following dialog window is shown Edit Spectrum Spectrum Name APAE abel eave Mn Drea eee AE Spectrum Color z Line Width Lo E Edit the spectrum and click OK How to edit the graph colors To customize the graph colors select the Edit Graph Color in the popup menu Set Graph Color Double Click to Edit Graph Color ltem Name Frame Background Graph Background Asis and Ticks Asie Test Measurement Text Gridline Zero Ans Legend Frame Legend Background Legend Text eee To change a color double click on the item and select a new color Edit Graph
6. displays a single m z channel eo ImageView ExpConc_110714 Slide2d_Tio30min10ug jpg_392_0 Conc Tiotropium 51 3 pmol mg The second image displays two different m z channels and how they are distributed in the tissue oe ImageView M24_DHB_23_2_2012MS_557 2 557 3_mask mslQuant 1 2 User Manual Version AB Page 35 of 39 Remove msIiQuant Files Remove msIQuant Files Remove mslQuant Files is a program that removes msIQuant generated files when they are not needed any longer It is also possible to remove redundant data when it is not needed any longer to save disk space E Remove msIQuant Files Select Spectra Top Folder That Contains msIQuant Files Spectra Top Folder C DATA TestSpectra msIQuant Files Bruker Files Remove Normalized CSV Files Remove Understructures A Remove xy dat Files Remove Complete Remove Folders Containing Average Files etc Progress Remove Spectra Processing files Spectra Processing generates xy dat files in every spectrum folder To remove these files when they are not needed any longer select the spectra top folder where they are contained check Remove xy dat Files and click on the Remove button The progress bar tells when the erase operation is complete Spectra Normalization generates normalized CSV files To remove these files when they are not needed any longer select the spectra top folder where they are contained check Remove Normalize
7. is not possible to set different values of x and y direction How to change the upper left position of an image Select Set Upper Left Position in the popup menu to change the upper left position It is possible to set a floating point of the position since the image is vectorized Set Upper Left Position Left Position 0 0 Upper Position 0 0 mslQuant 1 2 User Manual Version AB Page 33 of 39 Image View How to change the parameters of the color scale title and unit To change the value of the min and max intensity select the Set Min amp Max Intensity in the popup menu To return to the default intensities check the Set Default Intensities and click on OK button Set Min amp Max Intensity Min Intensity 1 000000 1 000000 Max Intensity 6 000000 6 000000 Set Default Intensities Set Title Intensity Set Unit It is also possible to change the title and the unit of the color scale How to save an image generated by Image View The present version of msiQuant has no export possibility of Image View The best way to save an image is to have the focus on Image View and to press lt Alt gt lt PrtScn gt to copy the image to the clipboard Then paste the image to the software where you want to show the image msIQuant 1 2 User Manual Version AB Page 34 of 39 Image View Examples of displaying a single or multiple m z channels Below are two examples of images The first image
8. 16 176804 250 0 154044 TiotropiumD3 M H 395 117306 300 0 118535 4 Name of Substance Substance Properties Recalibration Properties Substance Mass 9 O First order calibration Tolerance 0 In the recalibration dialog it is possible to open a calibration file that contains the calibrant needed to perform the recalibration The file extension for the calibration file is cal To create a new calibrant list just add the name of the substance in the Name of Substance edit window add the substance mass in Dalton and the tolerance in ppm Push the Add button to add the calibrant in the list It is possible to edit the list in two ways delete all substances by clicking the button Delete All or delete one substance by first select one and then click the button Remove When the list is created save the list with a unique name by pushing the Save As button Click the first or second order calibration and click the OK button Depending on which spectra processing steps to be done one need to check the different steps to be done The processing steps that can be selected are e Baseline correction e Baseline subtraction denoising e Smoothing e Peak picking e Recalibration mslQuant 1 2 User Manual Version AB Page 11 of 39 Spectra Processing Processing The different processing options are e Acquisition properties e Processing single fid file
9. 4 of 39 Spectra View Spectra View Spectra View is a program that displays mass spectra files It is capable to display following files e star dat mass spectra files e star csv mass spectra files e fid raw mass spectrum files that is created by Bruker Daltonics flexControl The format of the dat and csv files are text files were the first line can be a header that contains information like m z and Intensity The file must contain only two columns where the first column is the mass and the second column is the intensity The decimal separation must be a dot character value Ox2e and the column separation character can be a tab character value 0x09 space character value 0x20 or a comma character value Ox2c The line must end with carriage return character value OxOd and line feed character value Ox0a How to open mass spectrum files Since dat files are associated with Spectra View it will start Soectra View and open the file directly when double clicking on it in explorer Normally csv files are associated with Windows Excel but to open it from explorer right click on the csv mass spectrum file and click on Send To and select Spectra View that is in the menu To open a fid mass spectrum raw file in explorer right click on the fid file and select Open in the menu and select Spectra View in the Programs list It is also possible to start Spectra View and right clic
10. 5 How to zoom in and out and how tO MOVE SPectTa cccoocccccnccncnncnnnnncononarononarononononnnarononaronenarononanos 26 How to select a specific m z and intensity range or set axis and spectra offset ooccccccccccnnnccnncnona 27 How to measure m z and Oti as 27 DISPLIMOPUONAS A A A da 28 HOW LO delete or mde Specs de sateen tun anian Siwsatedevtuedeaameed beds 28 How to edit spectra names colors line Widths and Spectra Order ccccceeeccesesceeeseeneeseneetenseeees 28 HOwWwto CANA CS LO es 29 HOWTO CODY TAM E a8 Gch cto E A 30 How to copy the spectra within a Specific M Z range occcccccnnnnncnnnnnncnonnnanananaconnncnnnnccnnnnnono nono nonananass 30 HOW 1LO SelECt SPeCITIC SDECIia peakslanaa ds 30 HOW tO SCT peak DICKING Parameters ii us deca EN NAT 30 Mate VW ads ataca ica 31 HOWto Open image XMundo 31 How to select different interpolation Methods oocccnccnccnnnncnnccnnnnnnonnnonaronononaronnnnononnnnnnronnnonanonoss 32 FOw to change color Scale at A 33 Howto change the poesia didas 33 How to change the upper left position Of an iIMage cccoocccnncnnccnnnonnnonnnonaconnnnanonnnonononnnnnnconnonanononos 33 How to change the parameters of the color scale title and unit ooocccnnncnnnnncnnnnnannnnnannnnaccnnnnano 34 How to save an image generated by Image VIEW cccccssscccessecccsecceenceceusceeseueeeeeneeesescesaueceeseness 34 Examples of displaying a single or multiple M Z Channels
11. I_Quantitation_Workshop_msIQuant Data_300ctlTiss E Top Folder Create Quantitation Progress i When quantitation is made on experimental tissue it can be done to the entire tissue or on the ROI from the experimental tissue Select spectra folder for the experimental tissue if quantitation of the whole tissue shall be done However if quantitation of the ROI of the experiment the tissue shall be done select the spectra top folder and enable the checkbox Top Folder Then press the button Create Quantitation and the following dialog Create Quantitation of Tissue appears Create Quantitation of Tissue Data Parameters Export Intensity Export Concentration Data Quadratic Slope 0 0 Slope 138 9369269 Intercept 544 071655 Mass Filtering Parameters Region Parameters Center Mass 392 0997 Da Normalized Data per Pixel Mass Range 0 15 Da Use Maximum Intensity in Range 5 Integrate Intensity for Mass Range The default settings are the same as in the Create standard Curve dialog Click the OK button to generate the image xml file s For information The naming of the generated image xml file is ExpConc foldername center mass mass range H or A P or R H is when the peak intensity has been used and A when the peak area has been used P is when pixel by pixel is calculated and R when a whole region is calculated mslQuant 1 2 User Manual Version AB Page 22 of 39 XML
12. Mass Range Enable the checkbox Export Concentration Data and then click on the big button with a graph bitmap on and the next dialog Concentration Study will appear mslQuant 1 2 User Manual Version AB Page 19 of 39 Quantitation Tool Create Standard Curve Select Peak List File Peak List File uantitation_Workshop_msIQuant_Data_300ct Ctrl PeakList Peak_392 0997 csv m Mass m z 392 0997 Da Double Click on Region to Select a New Amount Remove Region Amount Peak Height Amount mg 0 000pmol 0 0 0 0 0 0 0 080pmol 0 08 129 28 2 5691565 0 160pmol 0 16 190 78 5 383129 0 320pmal 0 32 719 97 10 766259 0 640pmol a 1683 28 21 532518 1 300prnol le 5705 45 43 737926 2 600pmol 2 11470 37 87 475853 5 000pmol 5 22971 45 168 222794 Select Type for Slope Intercept Calculation 5 Use Amount 5 Use Amount mm Use Amount mg Spot Diameter 1 62 mm Slope 138 959269 Intercept 544071665 Tissue Density 1 03 Ri 0 997651 Tissue Thickness 14 0 um Tissue Volume am Polynomial Regression Tissue Mass Zero Intercept To find the extracted peak from the second step go to the top folder and the into the lt PeakList gt folder there is the extracted peak file e g the file lt Peak_281 2034 csv gt To make it easy to find the file the mass of the centroid peak is attached to the file name In the list box the regions from the concentration spots appear To edit the amount of added substance doubl
13. OX006Y056 0_ROOX006Y057 Spectra folder 2 50 ROOX010Y077 0_ROOX010Y078 0_ROOX010Y079 0_ROOX010Y080 0 _ROOX010Y081 Spectra folder 3 Spectra folder 4 The manual is also referring to spectrum folder Spectrum folders are those that are contained ina spectra folder e g folder 0 ROOXOO6Y053 is a spectrum folder When the data analysis with the program suite in MALDI Tools has been performed there are three new folders in the spectra top folder AVG PeakList and StDev folders DATA Spectra top folder Spectra folder 1 Spectra folder 2 Spectra folder 3 Spectra folder 4 QAVG PeakList StDev The AVG folder will contain the average spectra for each spectra folder The StDev folder will contain the standard deviation spectra for each spectra folder and a noise calculation for each average spectra that includes local noise and average spectrum noise The PeakList folder will contain the peak list for each average spectrum and extracted peak information file for a specific spectrum peak Pixel definition The definition of a pixel in this user manual is the same as sampling position where a mass spectrum is acquired Associated files List of file extensions and their associated programs e cal Mass spectrum recalibration file associated with Spectra Processing e par Labeled normalization parameter file associated with Spectra Normalization e dat Mass spectrum data file associated
14. Pixel analysis The program makes analysis of neighbor pixels how much they differ from each other with by Pixel Deviation Analysis and Pixel Ratio Calculation Below is a description of the algorithms Pixel Deviation Analysis The resolution does not matter when performing a pixel deviation analysis with quadratic pattern since the relative distance is always the same a al pa Pio lL Pro The algorithm works in the way that the distance to all eight e 4 o p neighbors is the same and is represented by the dotted circle L j o around the central pixel oe n Each pixel is named with x and y index p For each pixel there is an intensity also named with x and y index y The abbreviation of Pixel Deviation Analysis is for short PDA Differences for each neighbor pixel are calculated Dio lao z loo Do loi E loo D140 Lio loo Do 1 lo 1 loo except for the corner pixels where the differences are divided by the square root of 2 D11 l1 1 id loo V2 D11 L11 loo V2 D 1 1 l 1 1 id loo V2 D1 1 l1 1 id loo V2 The root mean square RMS is then calculated of the eight differences the PDA is equal to the RMS l 2 2 2 2 2 2 2 2 z Dk Dgn Dio Di Di D Di D PDA RMS Pixel Ratio Calculation The Pixel Ratio of a region of interest ROI is the average Intensity from the pixels divided by the average PDA of the pixels mslQuant 1 2 User Manual Version AB Page 2
15. a Processing Parameters Baseline correction with first quartile The baseline correction is an algorithm that straightens out the baseline when the baseline of a spectrum is drifting The method that is used to create a baseline is the simple moving first quartile SMQ1 The only parameter to set in this function is the length of the moving frame The value of this frame can be 0 20 of the total m z channels and the default value is set to 10 After creation of the baseline the baseline is subtracted with the spectrum To avoid negative spectrum values the modulus of the largest negative value is added to all intensities for all m z channels Baseline subtraction and baseline denoising Transform parameters The baseline subtraction and baseline denoising is performed with an algorithm called sorted mass spectrum transform SMST The noise part in the SMST transform is determined by three parameters Cutoff Cutoff intensity and Denoising level If the intensity is higher than the Cutoff intensity at the Cutoff of the SMST transform the transform is scaled down from O to the Cutoff to be equal as the set Cutoff intensity The peak part of the SMST transform is subtracted by a value so the transform fits the Cutoff intensity If the intensity is smaller at the Cutoff than the Cutoff intensity no processing is performed If a more aggressive denoising shall be done the Denoising level is set higher than 1 The derivative at the Cutoff is calculat
16. abeled ion file that contains the calibrant needed to perform the recalibration The file extension for the labeled ion file is par To create a new labeled ion list just add the name of the substance in the Name of Substance edit window add the analyte ion center mslQuant 1 2 User Manual Version AB Page 15 of 39 Spectra Normalization mass in Dalton and the mass range in Dalton Also add the labeled ion center mass in Dalton and the mass range in Dalton Push the Add button to add the labeled ion in the list It is possible to edit the list in two ways delete all substances by clicking the button Delete All or delete one substance by first select one and then click the button Remove When the list is created save the list with a unique name by pushing the Save As button Do not forget to check the labeled normalization button if a labeled normalization shall be performed If no other overall normalization is needed for the rest of the spectra like TIC Normalization just click No Normalization Click on the button Continue to perform the normalization and average spectra creation Match Spectra If regions of interest ROI have been defined and the spectra are located in a new top folder contained with the different ROI spectra folders one need to match these new spectra with the normalized spectra that has been performed in the first step This is done by first selecting the spectra sour
17. anual Version AB Page 8 of 39 Spectra Processing Spectra Processing is a program that performs general spectra processing The resulting spectrum is stored in each spectrum folder with a name called lt xy dat gt and is located in the same folder hierarchy as the lt fid gt file Below is an example of the hierarchy 0_ROOX011U062 1 1Ref pdata 6 acqu acqus fid 3 SpectraProcessing xml 3 sptype 3 xy dat The spectrum processing steps are e Baseline correction e Baseline subtraction and denoising e Spectrum smoothing e Peak picking and Recalibration mslQuant 1 2 User Manual E Spectra Processing Baseline correction with first quartile Baseline correction frame in of whole baseline 0 20 Baseline subtraction and denoising Transform parameters Cutoff 0 100 70 Cutoff intensity A U 0 5 Denoising level 1 10 1 Savitzky Golay filter for smoothing and differentiation First order Num of data points first order 3 9 Second order Num of data points second order 9 Noise calculation Noise cutoff 0 01 1 0 05 Peak Picking Peak picking SNR Recalibration Baseline correction Spectrum offset Da Baseline subtraction denoising 0 Peak picking Open calibration list Recalibration a E A E d 0 Aquisition properties Process single fid file Version AB Spectra Processing Spectra gt a b cin CSV Page 9 of 39 Spectr
18. ars by pressing lt Ctrl gt key on the keyboard It is then possible to zoom in and out the intensity axis by moving the scroll wheel on the mouse back and forth Sy If the Zoom in m z or intensity cursors appears and the spectrum mass spectrum is zoomed it is possible to move the spectrum in both m z and intensity direction by holding down the left mouse button and move the mouse on the spectrum area Once the spectrum starts to move the Hand cursor appears ts The Zoom in cursor appears by pressing the lt Alt gt key on the keyboard It is then possible E to zoom in the spectrum by selecting the zoom area with the mouse mslQuant 1 2 User Manual Version AB Page 26 of 39 Spectra View Re The Mass axis arrow cursor appears when holding the cursor over the m z axis Double click with the left mouse button on the m z axis to rescale to the full m z range If the mass spectrum is zoomed it is possible to move the spectrum in m z direction by holding down the left mouse button and move the mouse on the m Z axis Lett The Intensity axis arrow cursor appears when holding the cursor over the intensity axis Double click with the left mouse button on the intensity axis to rescale to the full intensity range If the mass spectrum is zoomed it is possible to move the spectrum in intensity direction by holding down the left mouse button and move the mouse on the intensity axis How to select a specific m z and intensity range or set axis and spectra
19. cccccnnonnnnnanananananona nana nanonononono nono nnnnnanos 35 Remove MSIQUANtE Fles nesnrni i tes ieee A A onwant seein E R EOR SEAT ele aeeided 36 Remove Spectra Processing Mesa di dicas 36 REMOVE Bruker FIGS add Oi E da sain dale Guid A 36 APPENA ADDF VIALIONNS a e ala E O 37 NUON CO ET o A A 38 mslQuant 1 2 User Manual Version AB Page 3 of 39 Legal and Regulatory Notices Legal and Regulatory Notices Copyright 2013 Biomolecular Imaging and Proteomics Group BIP Uppsala University All rights reserved All other trademarks are the sole property of their respective owners All Rights Reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Document History mslQuant 1 2 User Manual version AB November 2013 Warranty The information in this document is subject to change without notice BIP is not liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Use of Trademarks The names of actual companies and products mentioned herein may be the trademarks of their respective owners License for use and distribution mslQuant 1 2 is freeware software mslQuant 1 2 can be used on any computer including a computer in a commercial organization mslQuant 1 2 is developed in C with Microsoft VS2010 MFC and NET framework At present time the source co
20. ce folder that contains the e g control tissue spectra Second select the spectra destination top folder that contains the ROI Spectra Normalization Normalize Spectra Match Spectra Structure Create Structure Select Spectra Source Folder and Spectra Destination Folder Spectra Source Folder Spectra Dest Top Folder C DATA TestSpectra NewStructure Matching Normalized Spectra Between Destination and Source l Match Spectra Progress Information text Progress 100 0 Continue the spectra matching by clicking the button Match Spectra The normalized spectra are copied to the ROI spectra folders and an average spectrum is calculated for each ROI Copy Structure If data processing with several different normalization methods shall be done it is possible to copy the spectra structure mslQuant 1 2 User Manual Version AB Page 16 of 39 Spectra Normalization Spectra Normalization Copy Structure Create Structure Copy Structure Input spectra Source Folder Cu Copy Top Structure Copy Plain Structure Spectra Dest Folder ca Copy folder structure It is possible to copy a whole top structure or just a plain structure as a single spectrum folder The information that is copied is just folder structure not the underlying data Create Structure To create folder structures containing ROI and their spectra select first the spectra list from regions xml file created by Bruker
21. d CSV Files and click on the Remove button The progress bar tells when the erase operation is complete Spectra Normalization also generates the folders AVG StDev and PeakList To remove these folders with their content select the spectra top folder where they are contained check Remove Folders Containing Average Files etc and click on the Remove button The progress bar tells when the erase operation is complete Remove Bruker Files When ROI spectra are copied all information including raw files etc are copied as well This information is not needed when running msIQuant only the empty spectrum folders To reduce the disk space it is possible to remove this understructure To do this select the spectra top folder where they are contained check Remove Understructures and click on the Remove button The progress bar tells when the erase operation is complete When a copy of a whole experiment shall be erased it is possible to do this by selecting the spectra top folder where the experiment is contained check Remove Complete and click on the Remove button The progress bar tells when the erase operation is complete mslQuant 1 2 User Manual Version AB Page 36 of 39 Appendix Abbreviations Appendix Abbreviations Abbreviation CSV IMIS m z MALDI MS R PDA PRC RMS ROI RSD SD SMST SMQ1 SNR TIC TOF xml mslQuant 1 2 User Manual Explanation Com
22. de is not open source One doesn t need to register or pay for mslIQuant 1 2 but if the software is used for publication please cite the following article K llback P Shariatgorji M Nilsson A Andr n PE Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections J Proteomics 2012 Jul 27 Epub ahead of print doi 10 1016 j jorot 2012 07 034 mslQuant 1 2 User Manual Version AB Page 4 of 39 Protocol for Data Analysis Protocol for Data Analysis Conditions Version 1 2 of mslIQuant is only working together with Bruker Daltonics flexlmaging and the data format XMass If the new Container data format is used it must first be converted with flex Data Converter Further only full MS in reflectron or linear mode can be analyzed LIFT or FAST data can t be analyzed in current version of msiQuant Process flow The process flow below illustrates the overall protocol for the data analysis Vendor of MALDI IMS software Spectra View Define ROIs of calibration spots and experimental tissue Export the Display single spectra list from regions that contains information about the ROIs spectrum or multiple spectra Spectra Processing Perform spectra processing such as baseline correction denoising and subtraction spectra smoothing and recalibration Spectra Normalization Normalize spectra such as non normalization median TIC RMS or label
23. der polynomial by enable the check box Polynomial Regression This might be handy if low amounts of the substance are spotted and ion suppression is present If this option is selected the Zero Intercept cannot be selected in this version of the program When all settings are done click on the OK button and the settings will be transferred to the previous dialog Export Concentration Data Create Standard Curve Data Parameters E Export Intensity Export Concentration Data Quadratic Slope 0 0 Slope 133 969269 Intercept 544 071665 Mass Filtering Parameters Region Parameters Center Mass 392 0997 Da Normalized Data per Pixel MassRange 0 15 Da 2 Average Data per Region Use Maximum Intensity in Range 5 Integrate Intensity for Mass Range It is now possible to generate an image xml file for the concentration spots A selection that influences the image xml file is the Region Parameters If a uniform concentration is required per spot select Average Data per Region Otherwise leave as is Click the OK button to generate the image xml file mslQuant 1 2 User Manual Version AB Page 21 of 39 Quantitation Tool Create quantitation of tissue A Quantitation Tool Create Standard Curve Spectra Top Folder C DATA MSI_Quantitation_Workshop_msIQuant Data_300ctiCtrl Select m z of a Peak Create Standrad Curve Create Quantitation of Tissue Spectra Folder C DATA MS
24. ding After completed installation click the Finished button Shortcut icons are displayed on the desktop and in the start menu Note When Spectra Processing and Spectra Normalization is used for the first time look for the calibration list and or the parameter list in the folder lt C Program Files Biomolecular Imaging and Proteomics Group ProcessingParameters gt or in 64 bit operating system such as Windows 7 Installation re msIQuant InstallShield Wizard Ready to Install the Program The wizard is ready to begin installation If you want to review or change any of your installation settings click Back Click Cancel to exit the wizard C Program Files x86 Biomolecular Imaging and Proteomics Group msIQuant User Information Name MmsUser01 Company Microsoft InstallShield Y msIQuant InstallShield Wizard Installing msIQuant The program features you selected are being installed Please wait while the InstallShield Wizard installs msIQuant This may take several minutes SSE o InstallShield The InstallShield Wizard has successfully installed msIQuant Click Finish to exit the wizard Cancel lt C Program Files x86 Biomolecular Imaging and Proteomics Group ProcessingParameters gt To uninstall msIQuant go into Add or Remove Programs that is found in the control panel Search for mslQuant and then uninstall mslQuant 1 2 User M
25. e Process spectra 0 Aquisition properties Process single fid file Process spectra Acquisition properties To analyze the properties of a single pixel click the button Acquisition properties and select the fid file in the spectrum of interest Below is an example of the information that is put together Information Aquisition date and time 2011 07 14 19 54 27 796 Spot id lt ROOX007Y037 gt Motor pos X um 50625 Motor pos Y um 53640 Laser intensity 9 62 No of laser shots 500 No of m z channels 58753 Minimum m z 159 7566 Maximum m z 1000 1138 Minimum intensity 0 Maximum intiensity 65003 Processing single fid file To process a single spectrum click the button Process single fid file and select the fid file in the spectrum of interest All processing steps are stored as CSV files and can be viewed by Spectra View It is advisable to check the processed spectrum for some spots with Spectra View to confirm that the spectra processing is as expected One can view the file SpectraProcessing xml to check the numerical result from the different processing steps The generated files are e xy01 RawSpectrum csv Raw spectrum e xy02 Quartile1Spectrum csv Baseline of the raw spectrum e xy03 BaselineCorrSpectrum csv Corrected raw spectrum e xy04 TransformSpectrumNormMZ csv SMST of the corrected raw spectrum e xy05_TransformSpectrumNor
26. e click on the region name and edit the correct amount in the edit window that appears and enter the return key If one spots should be an outlier single click on the region and click on the button Remove The slope intercept and R for the standard curve are updated simultaneously There are three different selections that can be made to calculate the concentrations e The amount of substance alone e The amount per mm tissue e The amount per mg tissue Select the concentration option in the group box Select Type for Slope Intercept Calculation Depending on the selection measure the average spot diameter tissue thickness and tissue density For convenience the selected tissue volume and tissue mass will be calculated Tip If the whole spot is selected and the pixels in the spot are extracted count the pixels and multiply them with the x and y resolution to calculate the area The diameter from the average faa 7 area of the spots can be calculated by the formula d A i and is easier then predict the diameter by measuring the picture To view the standard curve click on the button with bitmap picture of a graph Below is an example of how it looks like mslQuant 1 2 User Manual Version AB Page 20 of 39 Quantitation Tool Ctrl 392 0997 Da y 136 97x 544 07 0 9977 To force the standard curve to go through origin enable the check box Zero Intercept There is also another option to form a 2 or
27. ed and is multiplied by the Denoising level A new Cutoff is determined where this calculated derivative is located and the denoising is performed as described above Savitzky Golay filter for smoothing and differentiation The spectrum smoothing is done with Savitzky Golay smoothing filter The smoothing method can be with either average or 2 degree polynomial The frame of data points must also be defined and must consist of an odd number The default value is set to smoothing of 2 degree polynomial with a frame of 9 data points Noise calculation The noise calculation is based on the SMST transform In this part the SMST transformed is the m z channels normalized from 0 to 1 The intensity is also normalized from O 1 The transform is then derived The parameter in the noise calculation Noise cutoff is the value of the derivative until the noise is considered to be localized The default value of the derivative Noise cutoff is set to 0 05 All values after this cutoff is set to zero and the SMST is retransformed The noise is then calculated as the root mean square RMS of all peaks in the noise Peak Picking The peak picking algorithm is calculated as the signal to noise ratio SNR and the default value is set to 2 The reason to do the peak picking is to be able to do recalibration of the spectrum The peak picking is done by deriving the spectrum When the derivative is crossing the x axis from positive to negative value there
28. ed normalization Create folder structures from the spectra list XML files and match normalized spectra with the structures This process will create average spectra from each ROI defined by the spectra list Quantitation Tool Select the ROI folder structure of the calibration spots Extract peak information from peaks of interest Create standard curve for quantitation The standard curve is then implemented to the normalized spectra of the experimental tissue An image xml file from the experimental tissue is created for further evaluation and display XML Evaluation Image View Evaluate the image xml file Following Display an image by the information Information is generated number of pixels from the image xml file average quantified value SD RSD min value max value pixel deviation analysis pixel ratio calculation mslQuant 1 2 User Manual Version AB Page 5 of 39 Protocol for Data Analysis Spectra folder definition To be able to use the programs in MALDI Tools the user has to understand the overall data structure There are two different expressions that are used spectra top folder and spectra folder In the spectra folder are all the spectra for regions of interest ROI or spectra from different experiments The spectra top folder though contains one or several spectra folders See the structure below a S3DATA Spectra top folder Spectra folder 1 0_ROOX006Y053 0_ROOX006Y054 0_ROOXO006Y055 0_RO
29. k on the window and the following popup menu appears Show Gridline Show Zero Axis Show Legend Show Baseline Open Spectrum Ctrl 0 Delete Spectrum Hide Spectrum Set m z and Intensity Range Ctrl R Edit Spectrum Name Color and Line Width Edit Graph Color Peak Option Copy Image Copy Spectra About SpectraView Quick Help Select Open Spectrum in the popup menu or just press lt Ctrl 0 gt to open a mass spectrum It is also possible to open several spectra with multiple selections Once a spectrum is opened it is possible to open one or several more spectra If spectra have been edited it is possible to reopen them again by selecting Recent Spectra in the system menu mslQuant 1 2 User Manual Version AB Page 25 of 39 Spectra View Spectra View Restore Move Size Minimize Oo Maximize x Close Alt Fa About Spectra View Quick Help Recent Spectra es Below is an example of how a mass spectrum is displayed O Spectra View mz 504 1068 Intensity 845 74 a u l i me A ard MUA Pa has tl 300 400 500 600 700 3900 How to zoom in and out and how to move spectra During the zoom operation different cursors appear and their specific zoom operation can take action When the Zoom in m z cursor appears it is possible to zoom in and out the m z axis by moving the scroll wheel on the mouse back and forth The Zoom in intensity cursor appe
30. mMZBaselineProc csv SMST with cutoff e xy06_TransformNormMZNormiInt csv SMST with cutoff and normalized intensity from O 1 e xy07_TransformSpectrum csv SMST with cutoff and the intensities corresponding masses e xy08 InvertedTransformSpectrum csv Retransformed SMST spectrum e xy09_GentleSmoothingSpectrum csv Spectrum smoothed with Savitzky Golay smoothing filter e xy10 DerivativeSpectrum csv Derivative of smoothed spectrum e xy11 SpectrumNoise csv Spectrum noise with removed peaks e xy12 DerivativeNoise csv Derivative of noise spectrum mslQuant 1 2 User Manual Version AB Page 12 of 39 Spectra Processing e xy13 PeakNoise csv Peak noise indication spectrum e xy14 Valleyindication csv Valley indication of smoothed spectrum e xy15 BaselineSmooth csv Baseline of smoothed spectrum e xy16 RecalibratedSpectrum csv Recalibrated spectrum e PeakList csv Peak list before recalibration e PeakListPost csv Peak list after recalibration e SpectraProcessing xml The result of the spectra processing Process spectra To process spectra in a top folder click the button Process spectra and select the top folder During this process a xy dat file and a SpectraProcessing xml file are created in each spectrum folder The progress bar will indicate how much of the spectra that have been processed mslQuant 1 2 User Manual Version AB Page 13 of 39 Spectra Normalization Spectra Nor
31. ma Separated Values Imaging Mass Spectrometry Mass to charge ratio Matrix Assisted Laser Desorption lonization Mass Spectrometry Coefficient of determination Pixel Deviation analysis Pixel Ratio Calculation Root Mean Square Region of Interest Relative Standard Deviation Standard Deviation Sorted Mass Spectrum Transform Simple Moving First Quartile Signal to Noise Ratio Total lon Current Time of Flight eXtensible Markup Language Version AB Page 37 of 39 Index A Acquisition properties Average spectra Average quantified value B Baseline denoising Baseline correction Baseline subtraction Bicubic interpolation Bilinear interpolation Bilinear Color Blend interpolation C Centroid mass Color scale Copy structure Create structure Cutoff Cutoff intensity D Denoising level Display mass spectra E Extracted peak F File extensions G Gridline Image xml file Installation of mslQuant Interpolation methods mslQuant 1 2 User Manual 12 15 23 10 10 10 32 32 32 19 20 30 32 16 17 10 10 10 25 19 28 18 23 31 7 32 L Labeled Normalization M Match spectra Median Normalization Measure m z and intensity N Nearest neighbor interpolation No Normalization Noise calculation p Peak height Peak mass Peak picking Pixel Pixel Deviation Analysis Pixel Ratio Calculation Pixel Size Process flow Process spectra Processing single fid file Q
32. malization is a program that Spectra Normalization Normalize spectra with different methods Makes average spectra from ROI and or tissue experiments Matches normalized spectra to later defined ROI Copies spectra structures when different normalization method is performed on same experiment Creates spectra structures from spectra list from regions Before running this program run Spectra Processing to create lt xy dat gt Normalize Spectra Spectra Normalization Normalize Spectra Match Spectra Copy Structure Create Structure Select Spectra Top Folder Spectra Top Folder AAURUANIES ae Select top folder normalize and then average regions Normalize Spectra Progress Progress After the spectra processing with Spectra Processing are performed normalization and creation of average spectra is done with Spectra Normalization Normalize Spectra When the normalization is performed the intensities are restored in respect to the normalization method for each spectrum Therefore must the experiments from the control tissue and the experiment tissue be normalized at the same time The spectra top folder must contain spectra folders for control tissue and experimental tissue When the button Normalize Spectra is pressed the following window appears mslQuant 1 2 User Manual Version AB Page 14 of 39 Spectra Normalization Normalization Method Normalization Method fo
33. might be a potential peak that is determined by the SNR The peak itself is calculated by applying a 3 degree polynomial of the spectrum where the derivative is crossing the x axis mslQuant 1 2 User Manual Version AB Page 10 of 39 Spectra Processing Recalibration When spectra are acquired during an MALDI imagine MS experiment they have to be recalibrated afterwards even if a calibration has been performed before an experiment This is because of how some MALDI MS instrument is constructed When a time of flight TOF MS is used the m z will differ because of the difference in tissue and matrix thickness The recalibration is performed against known peaks and the methods to this processing step are single calibrant recalibration or multiple calibrant recalibrations with 1 or 2 order To do a 1 order recalibration at least two known peaks are required For 2 order recalibration at least three known peaks are required For both 1 and 2 order recalibration a calibration curve is generated with best fit against the calibrant One point calibration can also be used if only one calibrant is in the list The one point calibration will only shift the peaks If a calibration before setting up an experiment has failed it is possible to correct a spectrum if a mass shift has occurred with up to 2 Da This value is set in the main dialog Recalibration Tiolung131024 Substance Substance Mass Da Tolerance ppm Tolerance Da Heme b 6
34. mslQuant 1 2 User Manual Evaluation and quantitation tool for MS Imaging Version AB Table of Contents Table of Contents Table or ECON tens ura 2 Fegalana Regulatory NOUICE S oe 4 Protocolfor Datar ANAS osea a a ven tee ase deaaasa oui isa nant aaandee teeayoasieatieeioagiae eacsels 5 CONCILIO Sr o ol 5 Process OWN tuscan A NN 5 Spectra folder definition csser E TE T E N oneneersoaeeee 6 PIXO CLOTITIE OU aaa 6 PSSOR ACC TOS te reste E E E S nce ue B nae ane eae A E meena eee ate cee 6 A o O 7 SECU POCOS OO 9 PIM Soo 10 Baseline correction with first QU a ci 10 Baseline subtraction and baseline denoising Transform parameteFrS oocccccnnccnnnonanonnnonanonnnnnaoos 10 Savitzky Golay filter for smoothing and differentiatiON occcconcncncnonnnonacnnnnananonananonarinonarononaso 10 Nore calculation ien rs 10 Peak PIN osa 10 e Oe EEE O 11 PROCESSING E naaa 12 ACQUISITION propertie Seder A ata 12 PROCESSING sinele TIC leia coito 12 PROCESS Dec radiata iasticds 13 Spectra NOM ZION e O OEI 14 Normalize Spectra ias 14 Mate Spectra a 16 CODY SUCUT di aaipa 16 Create SUE ds dll e do de a dieras de o dd kT UAC ATION TOO luso sas 18 Create standard CU 18 Create qua IAN USE vii 22 AME Ei WACO Nit dro tat eeutal space 23 Pero rea latinas 23 lA EP non a E A a ae tae et eeu eat 24 mslQuant 1 2 User Manual Version AB Page 2 of 39 Table of Contents Spectra VICW oein a No 25 Howtoopen mass spectrum THES ci a a 2
35. offset To set a specific m z and intensity range right click the right mouse button and click Set m z and intensity range in the popup menu or just press lt Ctrl R gt Set m z and Intensity Range Low m z 159 7566 Set Axis Offset 0 High m z 1000 1138 Set Spectra Offset 0 Set New Max Int 8176 16 Low Intensity 14 01 High Intensity 3925 6018 Cancel Set the values to get specific range of m z and intensity and click the OK button If the spectrum is close to the zero intensity it is possible to set a negative axis offset value to be able to lift up the spectrum If several spectra are displayed it is possible to separate them by setting the spectra offset If a peak is investigated and it is the highest in the whole spectrum it is possible to set a new maximum intensity to zoom out the spectrum How to measure m z and intensity The information about m z and intensity is shown all the time when the mouse is moved over the graph area It is also possible to measure the m z and intensity difference EN The Measure cursor appears when pressing lt Shift gt key on the keyboard and is used for measuring Am z and Aintensity Move the cursor to a specific peak press down the left mouse button and move the cursor to a new peak and release the mouse button A message box appears with the information about the Am z and Aintensity The information is automatically copied to the clipboard to be able to pas
36. p in creation of a standard curve is to select the top folder that contains the regions of interest ROI of the different spotted concentrations When this is done push the Create Peak List button This action will create peak lists of each average spectrum mslQuant 1 2 User Manual Version AB Page 18 of 39 Quantitation Tool The second step is to select the peak of interest by pushing the button Select m z of a Peak The following window appears Select m z of a Peak Select Single m z to Extract Mass m z Select Mass then push lt Extract Mass gt button Extract Single Mass Extract All Masses EN Click on the combo box button and a list of all peaks and their centroid mass and average peak height will appear Select the peak mass of interest and click on the button Extract Single Mass After peak extraction click the Exit button to close the window The third step is to create a standard curve based on the mono isotopic peak from the substance that is investigated Continue to click the button Create Standard Curve and the dialog Create Standard Curve appears Create Standard Curve Data Parameters E Export Intensity Quadratic Slope 0 0 Slope 100 0 Intercept 0 0 Mass Filtering Parameters Region Parameters Center Mass 800 0 Normalized Data per Pixel MassRange 0 15 Da 0 Average Data per Region 8 Use Maximum Intensity in Range 5 Integrate Intensity for
37. r Overall Spectrum 5 No Normalization TIC Normalization 5 Median Normalization 5 RMS Normalization Labeled Normalization for Selected Peaks Labeled Normalization The normalization methods for the overall spectrum are e No Normalization e TIC Normalization e Median Normalization e RMS Normalization Select one of the normalization methods by clicking one of the radio buttons The default selection is TIC Normalization After the normalization two new folders are created in the spectra top folder lt AVG gt and lt StDev gt The folder lt AVG gt contains average spectra and lt StDev gt the standard deviation spectra Labeled normalization method If a labeled substance is added during the tissue preparation one can normalize a peak in the spectrum against this labeled substance peak Up to 10 labeled substances can be labeled normalized in the same spectrum To add a list of labeled substances click the button settings and the following dialog appears Labeled Ion Analyte lon Pair for Normalization Substance A Mass Da A Range Da L Mass Da L Range Da TiotropiumD3H 392098476 0 15 395 117306 0 15 Name of Substance TiotropiumD3H Analyte Ion Labeled Ion Center Mass 392 098476 Da Center Mass 395 117306 Da Mass Range 0 15 Di Mass F E EN Di The interface is more or less the same as in the Recalibration dialog in the program Spectra Processing It is possible to open a l
38. rogram to display an image xml file generated from the Quantitation Tool The Image View is in its present state more or less a prototype of displaying images The goals with this software were to investigate rendering principle interpolation methods color scale implementation and overlay of up to three different images The image xml file is compatible with Bruker Daltonics flexlmaging and can imported as well The rendering technology is called Silk Screen rendering The bitmap image that is displayed is transferred from the original image that is vectorized How to open image xml files When the Image View has started a small image with 4x4 pixels is viewed The image comes from the article Bicubic interpolation from Wikipedia http Image View It is possible to open an image xml file in several different ways If the program is already open one can drag and drop an image xml file or open the image xml file from a popup menu by right click the window If the image xml file is listed in the explorer one can open the file by right clicking on the file and further click on Send To mslQuant 1 2 User Manual Version AB Page 31 of 39 Below is the popup menu displayed Open File Add File Convert Image to XML Interpolation Color scale gt Set Pixel Size Set Upper Left Position Set Min Max Intensities How to select different interpolation methods Image View The default in
39. te it directly to e g Microsoft Excel mslQuant 1 2 User Manual Version AB Page 27 of 39 Spectra View Display options The display options are Show Gridline Show Zero Axis Show Legend and Show baseline They can be all set in the popup menu When Show Gridline is set it displays the major gridline for both intensity and m z When Show Zero Axis is set it displays the zero intensity axis in the spectrum When Show Legend is set it displays the legend of the spectra The legend can be moved in the window but is set in default position when the window is resized Below is an example how the legend can look like 110714_Slide2d 0 ROOX006Y053 110714_Slide2d 0_ROOXO06 7054 110714 Slide2d 0 ROOXO06Y055 110714 Slide2d 0_ ROOXO06Y056 When Show baseline is set the baseline is displayed in the same color and width as its spectrum but with a dashed line How to delete or hide spectra When spectra are opened and if one wants to delete a spectrum or spectra select the Delete Spectrum from the popup menu Delete Spectrum Spectra Check Spectrum that Will be Deleted Spectrum Name O 110714 Slide2d Tio30mini0ug jpg_0_ROOXO06YO53 O 110714 Slide2d Tio30min1i0ug jpg_0_ROOXO06YO54 O 110714 Slide2d Tio30min10ug jpg_0_ROOXO06YO55 O 110714 Slide2d Tio30min10ug jpg_0_ROOXOO6YOS6 Check the spectrum that is going to be removed and click OK If one wants to hide one or more spectra select the
40. terpolation method of an image is nearest neighbor To change the method click on the Interpolation and select one of four interpolation methods Open File Add File Convert Image to XML Interpolation gt Y Nearest neighbor Color scale Bilinear Set Pixel Size ER Color Blend Set Upper Left Position wes Set Min Max Intensities Below is the four different interpolation methods displayed Nearest neighbor interpolation Bilinear interpolation Bilinear Color Blend interpolation Bicubic interpolation mslQuant 1 2 User Manual Version AB Page 32 of 39 Image View How to change color scale There are eleven different color scales to express the intensities for one m z channel all with linear scale Open File Add File Convert Image to XML Interpolation gt Color scale Black White Set Pixel Size Red Temperature Set Upper Left Position Thermal Y Blue Red Set Min Max Intensities Bruker Rainbow Biomap Rainbowl Biomap Rainbow2 Synapt Blue Red Yellow Red It is possible to view up to three different m z channels but only with red green and blue color scales How to change the pixel size Select Set Pixel Size in the popup menu to change the pixel size It is possible set a floating point as a pixel size since the image rendering is vectorized Set Pixel Size Pixel Size 3 0 In present version of the software a pixel is squared so it
41. with Spectra View mslQuant 1 2 User Manual Version AB Page 6 of 39 Installation Installation The installation of msIQuant is very simple and the whole package comes with a single self extracting file The installation file can be downloaded at www maldi ms The latest version of the installation is mslQuant 1 2 0 10 and the computer requirement is Windows operating system XP or higher After downloading the file current file name msIQuant_1 2 O 10 exe and store it on a place in the computer double click on the file and it will self extract A security warning dialog may appear since no valid digital signature is attached with the installation file When the self extracting file starts the a installation is prepared ls Preparing to Install msIQuant Setup is preparing the InstallShield Wizard which will guide you through the program setup process Please wait Decompressing msIQuant_1_2_0_10 msi A A welcome screen appears Click the Next gt JJ rnsiQuant InstallShield Wizard button to continue Welcome to the InstallShield Wizard for msIQuant The InstallShield R Wizard will install msIQuant on your computer To continue dick Next WARNING This program is protected by copyright law and international treaties mslQuant 1 2 User Manual Version AB Page 7 of 39 Current settings are displayed Start the installation by click on the Install button The installation is procee
Download Pdf Manuals
Related Search