1. Methodology for Molecular weight determination by MALDI instrumentation One of the application of MALDI is for the determination of the molecular weight of the peptide and protein, which can be done from the peptide mass fingerprint or from the spectrum  Related Los: Laser properties, Matrix properties > Prior Viewing – IDD-27. In gel digestion, IDD-29. Matrix preparation for MALDI analysis, IDD-30. Matrix Instrumentation > Future Viewing – IDD-31. MALDI-TOF data analysis, IDD-45. Proteomic Profiling Global  Course Name: Molecular weight determination by MALDI instrumentation  Level(UG/PG): PG  Author(s) : Dinesh Raghu, Vinayak Pachapur  Mentor: Dr. Sanjeeva Srivastava *The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license

2. Learning objectives After interacting with this learning object, the learner will be able to: 1. Operate the MALDI instrumentation 2. Analyze the mechanism behind the MALDI-MS analysis 3. Calculate the molecular weight of the protein 4. Assess the troubleshooting steps involved in the experiments. 5 3 2 4 1

3. Master Layout 5 3 2 4 1 Sample loading Parameters setting Firing of spots Spectrum analysis Slide 10-14 Slide 7 -9 Slide 6 Slide 5

4. Definitions and Keywords 5 3 2 4 1 1. Mass Spectrometry (MS): is a analytical technique used to measure intrinsic property of a bio-molecule, its mass, with very high dynamic sensitivity. 2. Matrix Assisted Laser Desorption Ionization (MALDI): MALDI is effective ionization system for generating gas-phase ions of proteins and peptides for mass spectrometric detection. The analyte of interest is embedded in dried matrix-sample is exposed to pulses from a UV laser for further separation and detection. 3. Spectrum data: is nothing but the collection of data in the mass to charge ratio plotted along the X-axis and its intensity on Y-axis.

5. Step 1: T1: Sample loading 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Take the user through the slides of IDD-29. Matrix preparation for MALDI analysis. Let user take the apomyoglobin sample as known standard for the analysis.

6. Step 2: T2: Parameter settings 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Take the user through the slides of IDD:30 MALDI instrumentation. Animate the steps from the same IDD till the firing step for sample. Let user take the sample as apomyoglobin as known standardand spot it on the MALDI plate.

7. Step 3: T3: Firing of spots 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Now let user select the actual target spot, by right-click on yellow region, and specifing the target spot and clicks on Fire button. Animate initially low peaks, now let user makes fine adjustment with 5 buttons, make movement in camera view also, now display more peaks with much high intensity. Let user keep playing around to complete 100 profiles. Now highlight Store button, after user click, let user have option to save the data. User must play around the spot region to find sweet spot, where the peaks are more in number with high intensity. After the 100profiles user can save the data.

8. Step 3: T3: Firing of spots 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Now user can select some other spot and start firing like in previous slide. In between firing, if user likes to pause, user hav a option like Abort, Resume. In case the peaks obtained are not fine, user can click on “abort” and click on “clear data” to clear all the data collected to re-start again. In between firing user have option for abort, resume, suspend and clear data. User can select these options depending on the profile data obtained.

9. Step 3: T3: Firing of spots 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity In most cases the default parameters for peak processing are best suited. If user is not okay with the data collected, user can make peak processing with the required settings. Instruct user to carry out peak processing. Let user select Processing> peak processing. Display peak processing with user controls. Let user have option for methos selection, baseline subraction, threshold, average display of profiles.

10. Step 4: T4: Spectrum analysis 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity After peak processing the data is ready to be saved and exported. Let user select File>Export>ASCII. A small window display for user control must pop up. Let the user selects the default values ad clicks on Save as.. to open the Save As window. Browse to your desired location, enter a File name, and click ‘Save’. The PMF data will be saved. Once the PMF data is ready, data in the excel format can be exported, and saved.

11. Step 4: T4: Spectrum analysis 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Instruct user to calculate the molecular mass. let user select any two peaks. For example in the figure highlight x1 and x2. let user make a note of it. The mass can be calculated from any two peaks by taking the difference and applying the formulas.

12. Step 4: T4: Spectrum analysis 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Instruct user to calculate the molecular mass. Display the above 4 equation. Now that user knows x1, x2 and mH is 1, user can calculate the value for Z by equation 3. now all the values must be put in equation 4 to calculate the value for M. Animate the steps. M:molecular mass of the analyte molecule. x1: m/z ratio of the analyte molecule with z protons. X2: m/z ratio of the analyte molecule wit (z+1) protons. mH: mass of hydraogen ataom

13. Step 4: T4: Spectrum analysis 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Instruct user to calculate the molecular mass. With the values of x1=998.2 and x2=942.8. deduce the calculation of mass for apomyoglobin. The mlecular mass of apomyoglobin is around 16951.49u. This can be cross checked by doing the data analysis through MASCOT serach. mH: mass of hydraogen ataom

14. Step 4: T4: Spectrum analysis 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity In similar way molecular mass of a given spectrum of a particular protein can be calculated easily. In case of confirmation the same spectrum can be analysed through the MASCOT engine. For more information please go through future viewing IDD. In similar way provide few x1 and x2, or let user choose few peaks and their values to calculate the molecular mass.

15. Animation area In slide-14: if user is unable to find the Molecular weight from the spectrum. Instruction: let user note down the peaks and carry out MASCOT search to get the Molecular weight from the database. Instructions/ Working area Credits Name of the section/stage Interactivity area Tab 01Tab 02Tab 03Tab 04Tab 05Tab 06 Button 01 Button 02 Button 03 Introduction Slide 10-14 Slide 7 -9 Slide 6 Slide 1-4 Slide 5

16. Questionnaire: APPENDIX 1 Question 1 The moleclar weight of apomyoglobin? a) 16kDa b) 10kDa c) 11kDa d) 5kDa Answer : a) 16kDa Question 2 In time of flight, analytes are separated depending upon? a)Distance travelled b)Velocity of alalyte c)Mass d)Non of above Answer:c) Mass

17. Questionnaire: APPENDIX 1 Question 3 For higher molecular weight proteins, separation is best in? a)Reflectron mode b)Linear mode c)Acquisition mode d)Laser mode Answer : a) Reflectron mode Question 4 Which of the following is not a mass analyzer? a) Time-of-Flight (TOF) b) Quadrupole (Q) c) Ion traps d) MALDI Answer: d) MALDI

18. Questionnaire: APPENDIX 1 Question 5 The wavelength of laser used for ionization? a) 337nm b) 437nm c) 537nm d) 637nm Answer: a) 337nm 6. Question 5 MALDI is a a) Soft ionization technique b) Hard ionization technique c) Both of them d) None of them Answer a) Soft ionization technique

19. Links for further reading Papers: 1.Hillenkamp F, Karas M, Beavis RC, Chait BT. Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal Chem. 1991, 63, 1193A-1203A. 2. Karas M, Glückmann M, Schäfer J. Ionization in matrix- assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors. J Mass Spectrom. 2000, 35, 1-12. 3. Hillenkamp F, Karas M. Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization. Methods Enzymol. 1990, 193, 280-295. Books:  Proteomics: A cold spring harbor laboratory course manual by Andrew J L and Joshua L, 2009. APPENDIX 2

20. Summary APPENDIX 3 MALDI-MS instrument helps to determine or calculate molecular weight of an protein by just going through the spectrum data. The rough estimate from the spectrum data for molecular weight determination helps user to save lot of time and carry out further analysis by selecting each peak properly.