This webinar series demonstrates the use of the Thermo Freestyle 1.8 SP2 QF1 software for processing LCMS product-ion mass spectra &nbsp;with the NIST 23 Mass Spectral Search Program (v.3.0). &nbsp;Spectra are easily exported from Freestyle to NIST Search for component identifications utilizing the NIST MSMS libraries and any other available user, crowd-sourced, and commercial MSMS libraries. &nbsp;Freestyle is a very convenient software package for processing data obtained with the their Orbitrap LCMS instruments. &nbsp;In one run both the MS1 and MS2 spectra are obtained in both positive and negative ion modes. &nbsp;The Diode arrary and UV channels can also be easily displayed for targeting the desired species noted in LC analyses.<h5>Handout for whole series</h5><ul><li>Part 1: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Part_1_Data_Processing_My_Favorite_e29bfc0816.pdf">My Favorite Processing Mode in Freestyle</a></li><li>Part 1A: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Part_1_A_Data_Processing_Preferred_plus_Nearby_Precursors_33bc95778b.pdf">Using "Nearby Precursor" Enhanced Processing Mode in FreeStyle</a></li><li>Part 2: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Part_2_Data_Processing_Alternate_99a4b51fa6.pdf">Alternate Processing Modes in Freestyle</a></li><li>Part 3: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/part_3_improved_handout_d14f81cc32.pdf">Setup of Basic Settings in Freestyle and NIST Search</a></li><li>Part 4: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Part_4_NIST_search_type_demonstration_adb3cdc1c5.pdf">Different NIST MSMS Searches Demonstrated</a></li><li>Part 5: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Part_5_Elemental_Composition_demonstration_27e151914b.pdf">Elemental Processing within Thermo Freestyle</a></li></ul><h3>Part 1: Data Processing for NIST MSMS Search Using Thermo Freestyle Software (My Favorite Approach)</h3>This guide outlines my preferred method for processing data in Thermo Freestyle software for NIST MSMS searches. These steps streamline data parsing and ensure accurate mass spectral interpretation for LC-MS and MSMS analyses.<h4>Equipment &amp; Software</h4><ul data-spread="false"><li>Thermo Orbitrap MSMS</li><li>Thermo Freestyle Software</li><li>NIST 2023 Tandem Library</li></ul><h4>Orbitrap Data One Acquisition</h4><ul data-spread="false"><li>LC-MS and MSMS Analyses</li><li>Non-targeted acquisition of MSMS</li><li>Positive and Negative Mode</li><li>Photo Detector Array (PDA) and UV-VIS Analyses</li><li>Example compounds:<ul data-spread="false"><li>Uvinul 3000 (MW 214)</li><li>Tinuvin 234 (MW 447)</li></ul></li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_NIST_2023_Tandem_Library_Great_Resource_for_Unknown_Identifications_f4fc13b103.jpg" alt="James Little: NIST 2023 Tandem Library Great Resource for Unknown Identifications" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_NIST_2023_Tandem_Library_Great_Resource_for_Unknown_Identifications_f4fc13b103.jpg 245w," sizes="100vw" width="1384" height="854"><h4>My Favorite Data Processing Workflow</h4><ul><li>shared with me by colleague</li></ul><h5>Step 1: Setting Up Freestyle Windows</h5>To optimize the workspace for data processing:<ol data-spread="false"><li>Open three windows at the bottom of Freestyle:<ul data-spread="false"><li>Top window: Positive ion MS1</li><li>Second window: Negative ion MS2</li><li>Bottom window: UV or PDA trace</li></ul></li><li>Save this layout in Workspace Options/Layouts to ensure automatic parsing when opening new files.</li><li>Add the Nearby Precursors feature to simplify MSMS selection (except for averaged MS spectra).</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Step_1_Setting_Up_Freestyle_Windows_0dac4585f9.jpg" alt="James Little: Step 1 - Setting Up Freestyle Windows" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Step_1_Setting_Up_Freestyle_Windows_0dac4585f9.jpg 245w," sizes="100vw" width="1839" height="403"><h5>Step 2: Adjusting the Window Layout</h5><ol data-spread="false"><li>Use Select Filter to:<ul data-spread="false"><li>Change the top window to positive ion</li><li>Change the second window to negative ion</li><li>Choose PDA or UV for the bottom window</li></ul></li><li>The spectrum window will display the selected MS/MS data for further processing.</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Step_2_Adjusting_the_Window_Layout_d18baf8c37.jpg" alt="James Little: Step 2 - Adjusting the Window Layout" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Step_2_Adjusting_the_Window_Layout_d18baf8c37.jpg 245w," sizes="100vw" width="1829" height="1042"><h5>Step 3: Processing MSMS Data</h5><ol data-spread="false"><li>Left-click in the top box for the positive ion chromatogram.</li><li>Right-click to average the spectrum in the Spectrum box.</li><li>Subtract background if necessary before averaging.</li><li>Populate the Avg Range box and select Range 1.</li><li>Go to Positive Ion Chromatogram, specify the range, and confirm.</li><li>Avoid averaging MS2 spectra when exporting to NIST search, as it will convert accurate mass to nominal mass.</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Step_3_Processing_MSMS_Data_5a5e2b87b5.jpg" alt="James Little: Step 3 - Processing MSMS Data" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Step_3_Processing_MSMS_Data_5a5e2b87b5.jpg 245w," sizes="100vw" width="1878" height="1059"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Step_3_Processing_MSMS_Data_1_131f4645ca.jpg" alt="James Little: Step 3 - Processing MSMS Data" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Step_3_Processing_MSMS_Data_1_131f4645ca.jpg 234w," sizes="100vw" width="1513" height="1011"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Step_3_Processing_MSMS_Data_2_6ff9040f00.jpg" alt="James Little: Step 3 - Processing MSMS Data" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Step_3_Processing_MSMS_Data_2_6ff9040f00.jpg 245w," sizes="100vw" width="1738" height="1046"><h5>Step 4: Selecting an MS2 Spectrum for NIST Search</h5><ol data-spread="false"><li>Identify an m/z value of interest (e.g., m/z 215.0702).</li><li>Apply Chromatogram range filter to isolate the target ion.</li><li>The MS2 spectrum will replace the MS1 chromatogram in the top window.</li><li>Click on the peak of interest to generate an MS2 spectrum.</li><li>Repeat the process for Negative Ion MS2 (MSMS) spectrum.</li><li>Send spectra to NIST MS search for compound identification.</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Step_4_Selecting_an_MS_2_Spectrum_for_NIST_Search_271afd5538.jpg" alt="James Little: Step 4 - Selecting an MS2 Spectrum for NIST Search" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Step_4_Selecting_an_MS_2_Spectrum_for_NIST_Search_271afd5538.jpg 245w," sizes="100vw" width="1849" height="1125"><h3>Conclusion</h3>Using this workflow, Thermo Freestyle software efficiently processes LC-MS/MS data for NIST searches, ensuring accurate identification of unknown compounds. By saving custom layouts and avoiding averaged MS2 spectra, this method maximizes precision and efficiency.

 Data Processing for NIST MSMS Search Using Thermo Freestyle Software (My Favorite Approach)

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Prof. Helen Cooper, Ph.D., an expert analytical chemist from the University of Birmingham, recently discussed the latest advancements in imaging large multiprotein complexes using native Nano-spray desorption electrospray ionization (nano-DESI)<a> </a>mass spectrometry during a webinar<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5055"> View on-demand webinar</a>.<h3>Background on native ambient mass spectrometry imaging</h3>Native ambient mass spectrometry imaging allows scientists to visualize the spatial distribution of proteins and their complexes in thin tissue sections. This technology provides valuable insights into protein therapeutics, such as monoclonal antibodies. However, analyzing large multiprotein complexes using existing imaging technologies remains challenging.Dr. Cooper discussed improvements in mass spectrometry imaging that enable the characterization of precise proteins present in multiprotein complexes. During this webinar, you will learn about the Cooper Lab’s experience using the Thermo Scientific Orbitrap Eclipse tribrid mass spectrometer and the<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/748"> Thermo Scientific Orbitrap Ascend</a> structural biology tribrid mass spectrometer.<h3>Key topics covered</h3><ol><li>Orbitrap Ascend structural biology tribrid mass spectrometer: Learn how this instrument can accelerate native workflows with its capabilities for isolating ions up to m/z 8,000 in the quadrupole and detecting up to m/z 16,000 in the Orbitrap mass analyzer.</li><li>Deriving cellular information: Understand how native mass spectrometry of protein complexes can provide cellular information.</li><li>Combining imaging MS with native structural analysis: Discover how this combination can be used to provide proteoform-level information.</li></ol><h3>Presentation overview</h3>The presentation begins by explaining the goal of combining native mass spectrometry and mass spectrometry imaging to obtain simultaneous information about spatial distribution and protein structure directly from tissue. This approach integrates three strands of mass spectrometry:<ol><li>Native mass spectrometry: Proteins are electrosprayed from non-denaturing conditions, retaining non-covalent interactions in the gas phase, keeping protein assemblies intact.</li><li>Top-down mass spectrometry: Protein ions are fragmented to provide sequence information, identifying the protein and post-translational modifications.</li><li>Ambient mass spectrometry imaging: Allows spatial analysis of proteins in thin tissue sections. Performing native nano DESI imaging on tissue sections involves using a liquid junction sampling technique. This method allows them to sample tissues without disrupting protein structures.</li></ol><h3>Case study: Metal-deficient SOD1 dimer in ALS</h3>A significant case study involved imaging the metal-deficient SOD1 dimer in spinal cord tissue from a mouse model of amyotrophic lateral sclerosis (ALS). The study revealed an abundance of metal-deficient SOD1 dimer in regions of pathology, which correlates with ALS pathology. This was achieved using the Orbitrap Eclipse Tribrid Mass Spectrometer.<h3>Instrumentation and achievements</h3>Practical molecular weight limitsThe Orbitrap Eclipse Tribrid Mass Spectrometer has been instrumental in achieving significant milestones:<ul><li>Identification of the largest protein assembly from tissue at 145 kilodaltons.</li><li>Imaging of a 113 kilodalton aquaporin 0 tetramer.</li></ul>Proton transfer charge reduction (PTCR)PTCR has been utilized to determine intact masses and improve sensitivity. This technique allows the separation and identification of protein species from complex spectra, addressing the challenges of working with low-abundance signals and complex mass spectra.<h3>Advancements with Orbitrap Ascend structural biology MS</h3>Data independent acquisition (DIA)-PTCRThe DIA- PTCR method involves scanning along the m/z range and subjecting slices to PTCR. This method has enabled the detection of approximately 100 proteoforms, providing comprehensive proteoform discovery.<h3>Imaging higher molecular weight proteins</h3>The Orbitrap Ascend structural biology tribrid MS led to the imaging of higher molecular weight proteins, such as the 231 kilodalton pyruvate kinase tetramer. This represents a significant improvement, more than doubling the previous molecular weight range for mass spectrometry imaging of proteins.<h3>Conclusion</h3>Native ambient mass spectrometry imaging is a comprehensive platform for in situ native protein characterization and imaging. The advancements provided by the Orbitrap Ascend structural biology tribrid mass spectrometer have enabled significant progress in this field, doubling the molecular weight range for imaging and facilitating the characterization of larger macromolecular machines.This progress highlights the potential for deeper insights into protein structures and their functions within biological tissues, paving the way for advancements in understanding complex biological systems and developing new therapeutic strategies.<blockquote>Learn more at <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms/orbitrap-tribrid-mass-spectrometers/orbitrap-ascend-tribrid-mass-spectrometer/systems/orbitrap-ascend-structural-biology-tribrid-ms.html">www.thermofisher.com/OrbitrapAscendStructuralBiology</a>Visit us on <a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/">LinkedIn:</a> #StructuralBiology #NativeMS #nanoDESI #OrbitrapAscendStructuralBiology</blockquote>

Imaging Large Multiprotein Complexes Using Native Nano DESI Mass Spectrometry

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