<h3><strong>About us</strong></h3>
<p>Our laboratory is equipped with high-end mass spectrometry instruments, which offers services in field of <strong>PROTEOMICS</strong> and <strong>METABOLOMICS</strong>. All our services are available for everybody from the academic and non-academic areas.</p>
<h5><a href="https://www.biocev.eu/en/services/omics-mass-spectrometry-core-facility.6#core-facility-services"><strong>Our Services:</strong></a></h5>
<ul>
<li><strong>Preparation of samples</strong> for analysis (cells, body fluids, tissues, plants etc.)</li>
<li><strong>Targeted</strong> or <strong>untargeted</strong> <strong>proteomics</strong> and <strong>metabolomics</strong> analysis</li>
<li><strong>Data analysis</strong> and basic statistical evaluation</li>
<li><strong>Consultation</strong> of experimental design and the results</li>
</ul>
<h5><strong>Proteomics:</strong></h5>
<p>We are modern well equipped laboratory with the technical background able to perform most of the currently used <a href="https://www.biocev.eu/en/services/omics-mass-spectrometry-core-facility.6/untargeted-proteomics.24?t=s"><strong>untargeted</strong></a> and <a href="https://www.biocev.eu/en/services/omics-mass-spectrometry-core-facility.6/targeted-proteomics.25?t=s"><strong>targeted</strong></a> proteomic measurements. We provide full proteomics service, including protein isolation from the supplied sample to evaluation and consultation of the measured data. Since 2015, we handle approximately 300 orders per year (roughly 3000 samples). Results based on measurements from our laboratory were used in dozens of publications. To consult your plan, please contact Karel Harant (karel.harant@natur.cuni.cz) or Pavla Talacko (pavel.talacko@natur.cuni.cz).</p>
<h5><strong>Metabolomics:</strong></h5>
<p>As a service, we provide <a href="https://www.biocev.eu/en/services/omics-mass-spectrometry-core-facility.6/targeted-metabolomics.26?t=s"><strong>targeted</strong></a> and <a href="https://www.biocev.eu/en/services/omics-mass-spectrometry-core-facility.6/untargeted-metabolomics.30?t=s"><strong>untargeted</strong></a> analysis of small molecules. Thanks to the combination of equipment for <strong>LC-MS</strong> and <strong>GCxGC-MS</strong>, it is possible to measure a wide range of small molecules. We use several types of <strong>C18</strong> and <strong>HILIC</strong> stationary phase columns on LC. For GC, we have various columns for different types of analytes. Individual measuremnts must always be consulted in advance, an individual approach and method must be chosen for each group of molecules. Please contact Olga Součková (souckoo@natur.cuni.cz) for LC-MS or Petr Žáček (zacek@natur.cuni.cz) for GCxGC-MS.</p>
<blockquote>
<p><strong>All our services are available for everybody from the academic and non-academic areas.</strong></p>
</blockquote>
<blockquote>
<p><strong>Contact us before you start your first experiment: +420 325 873 925!</strong></p>
</blockquote>
<blockquote>
<p><strong>23.7.2024 Waiting time from “sample to result” is approximately 6-8 weeks.</strong></p>
</blockquote>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_cover_image_7cb6de4462.jpg" alt="BIOCEV: BIOCEV OMICS Mass Spectrometry Core Facility"/></p>
<h3><strong>Services</strong></h3>
<ul>
<li>Preparation of samples for analysis</li>
<li>Untargeted and targeted proteomics</li>
<li>Untargeted and targeted metabolomics</li>
<li>Data analysis and basic statistical processing</li>
<li>Consultation of experiment design and measured data</li>
</ul>
<h4><strong>Untargeted proteomics</strong></h4>
<h5><strong>Up to 8000 identified proteins in one injection.</strong></h5>
<p>A basic type of proteomic experiment. Complete lysate is digested with trypsin. The resulting peptides are desalted and separated on a reverse phase column. Due to the low amount of proteins and the high complexity of the sample, liquid chromatography is currently most often used in nanoflow - hundreds of nanoliters per minute. Liquid chromatography is directly connected to the electrospray of the Orbitrap Fusion or Orbitrap Ascend mass spectrometer. The mass spectrometer measures alternately in two modes: MS¹, where peptide masses are measured in the Orbitrap with high resolution and accuracy, and MS², in which peptides selected from the previous MS¹ scan are fragmented. These are measured either again in the Orbitrap or in the ion trap. The ion trap provides lower accuracy and resolution than the Orbitrap, but is faster and more sensitive.</p>
<blockquote>
<p><strong>We offer untargeted proteomics measurement and analysis of post-translational modifications in different types of samples (cells, tissues, body fluids, plant samples, etc.).</strong></p>
</blockquote>
<blockquote>
<p><strong>Our newest service is &quot;Thermal proteome profiling (TPP)&quot; that allows you direct detection of drug-protein interactions in living cells.</strong></p>
</blockquote>
<h5><strong>Examples of analyses:</strong></h5>
<ul>
<li>LFQ (label free quantification)</li>
<li>TMT (tandem mass tags)</li>
<li>TPP (Thermal proteome profiling)</li>
<li>phosphoproteome profiling</li>
<li>pull-down experiments</li>
</ul>
<h5><strong>Tips for sample handling:</strong></h5>
<ul>
<li>do not use trypsin for cell harvesting</li>
<li>inform us about presence of detergent</li>
<li>always keep in mind same protein loading for pull-down experiments (eg. Co-immunoprecipitation)</li>
<li>do not use phosphatase inhibitors for phosphoproteomic experiments</li>
</ul>
<h4><strong>Targeted proteomics</strong></h4>
<h5><strong>Accurate quantification of selected proteins.</strong></h5>
<p>Method aims to validate the results from an untargeted approach. It is performed on a triple quadrupole in SRM mode or using the MRM method on an Orbitrap. It is an an alternative approach to common techniques such as western blot. The advantage of this approach is the possibility to analyze several proteins (dozens) at once during one injection. This eliminates the problem of finding suitable antibodies.</p>
<blockquote>
<p><strong>The targeted proteomics service is always project specific and we offer it after prior meeting with the customer.</strong></p>
</blockquote>
<p>Parallel analysis of peptides labeled with heavy amino acids is essential. These serve as an internal standards.</p>
<p>The amount of proteins that can be targeted in one injection using a triple quadrupole, is strongly dependent on the concentration of the target proteins in the sample. The lower the expression of the target proteins, the less it is possible to analyze them in one injection due to the need for higher loading times. Information of fragmentation properties of targeted peptides is also important. This is usually obtained from an untargeted experiment, which should precede the targeted proteomic analysis. If the protein of interest was not detected during an untargeted experiment, then its targeted analysis is very difficult. However, in some cases fragmentation information can be obtained from publicly available databases.</p>
<h5><strong>Tips for sample handling:</strong></h5>
<ul>
<li>do not use trypsin for cell harvesting</li>
<li>inform us about presence of detergent</li>
<li>always keep in mind same protein loading for pull-down experiments (eg. Co-immunoprecipitation)</li>
<li>do not use phosphatase inhibitors for phosphoproteomic experiments</li>
</ul>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_Targeted_proteomics_1_f0605d8efc.jpg" alt="BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Targeted proteomics."/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_Targeted_proteomics_2_f1af260d27.jpg" alt="BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Targeted proteomics."/></p>
<h4><strong>Untargeted metabolomics</strong></h4>
<p>The goal of untargeted metabolomics is to cover the widest possible spectrum of metabolites present in the sample, their identification, and quantification. The development of this approach was made possible by the recent advances in instrumentation and computing. Our laboratory equipment enables non-targeted analyses by liquid and gas chromatography.</p>
<blockquote>
<p><strong>We offer untargeted analyses of metabolites in various biological matrices and basic statistical evaluation of the data (Figure 2, 3, 4).</strong></p>
</blockquote>
<blockquote>
<p><strong>Our newest service is stable isotope tracing (Figure 1).</strong></p>
</blockquote>
<p>HPLC-MS/MS (Figure 2): Analyzes are performed on Thermo Scientific equipment on an Ultimate 3000 liquid chromatograph coupled to an Orbitrap Fusion mass detector. The measured data are evaluated by the Compound Discoverer software.</p>
<p>GCxGC-MS (Figure 3): Analyzes are performed on a two-dimensional comprehensive chromatograph connected to a Pegasus 4D mass detector. We evaluate the data in ChromaTOF v4.5 software.</p>
<p>The liquid chromatography method covers non-volatile substances, generally polar, soluble in water, and polar organic solvents (methanol, acetonitrile...). The gas chromatography method is more suitable for analyzing stable volatile non-polar substances up to a weight of approx. 1000 Da. The range of measured substances can be increased for both methods eg., by chemical modifications (derivatization) of the analyzed molecules, which adjust their polarity and volatility (for GC) and/or stability (for LC). We choose a suitable technique or a combination of several techniques based on prior consultation with the customer.</p>
<p>The untargeted analysis results in a list of substances found in the samples, a proposal of probable structures, and an indication of the quantity of individual compounds. We also provide a basic statistical evaluation focusing on looking for differences between the tested sample groups (Figure 4).</p>
<h5><strong>Tips for sample handling:</strong></h5>
<ul>
<li>do not use phosphate buffers in the sample</li>
<li>inform us about sample type, growth medium, chemicals used, etc.</li>
</ul>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_Untargeted_metabolomics_Fig_1_11750e6992.jpg" alt="BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 1."/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_Untargeted_metabolomics_Fig_2_5ca1521705.jpg" alt="BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 2."/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_Untargeted_metabolomics_Fig_3_f4f1050fc5.jpg" alt="BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 3."/></p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_Untargeted_metabolomics_Fig_4_b3e198dbc9.jpg" alt="BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 4."/></p>
<h4><strong>Targeted metabolomics</strong></h4>
<p>Targeted metabolomics includes analyses of selected metabolites, which are then detected selectively and with greater sensitivity than applying an untargeted approach. It is also possible to quantify substances both relatively and absolutely. In the case of absolute quantification, it is necessary to supply a suitable labeled analog of the determined substance.</p>
<blockquote>
<p><strong>We offer analysis of primary cellular metabolites - see List of substances.</strong></p>
</blockquote>
<blockquote>
<p><strong>We analyze metabolites in tissues, cell pellets, incubation media, and body fluids (plasma, urine...).</strong></p>
</blockquote>
<blockquote>
<p><strong>We also offer the possibility of developing a method for the analysis of substances that are not in the attached list or for other matrices.</strong></p>
</blockquote>
<p>The attached list contains substances with the analysis of which we have experience. We adapt each method to the type of experiment and matrix. We can develop a method for any substance or group of substances capable of being detected by liquid and gas chromatography techniques (both connected to a mass detector or a UV detector in liquid chromatography). In the case of the development of a new method, it is necessary to supply the standards of these substances.</p>
<h5><strong>Tips for sample handling:</strong></h5>
<ul>
<li>do not use phosphate buffers in the sample</li>
<li>inform us about sample type, growth medium, chemicals used, etc.</li>
</ul>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/BIOCEV_Introducing_BIOCEV_OMICS_Mass_Spectrometry_Core_Facility_Targeted_metabolomics_List_of_substances_a6a4198b34.jpg" alt="BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Targeted metabolomics: List of substances."/></p>
<h3><a href="https://www.biocev.eu/en/services/omics-mass-spectrometry-core-facility.6#core-facility-equipment"><strong>Equipment</strong></a></h3>
<h4><strong>Mass spectrometers</strong></h4>
<h5><strong>Orbitrap Ascend</strong></h5>
<ul>
<li><a href="https://lcms.labrulez.com/library?instrumentationItems=31-32-33-30&amp;mainauthorItems=3&amp;search=Ascend"><strong>Application in the LabRulezLCMS library</strong></a></li>
</ul>
<p>Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer, offering new capabilities for multiplexed proteomics and native protein characterization. The mass spectrometer enables improved sample throughput, versatility and ease-of-use — producing high quality data with simplicity. More samples can be analyzed at lower concentrations, enabling faster, more sensitive analysis, and macromolecule experiments, that were not possible before, can now be achieved. The new Auto-Ready ion source for calibration maximizes convenience, allowing the user to spend more time on the results, and less time on instrument set-up.</p>
<h5><strong>Orbitrap Fusion</strong></h5>
<ul>
<li><a href="https://lcms.labrulez.com/library?instrumentationItems=31-32-33-30&amp;mainauthorItems=3&amp;search=Fusion"><strong>Application in the LabRulezLCMS library</strong></a></li>
</ul>
<p>Mass spectrometer combining Quadrupole, Ion trap and Orbitrap. The quadrupole is used as a mass filter for the selection of precursors, after which the instrument branches and the ions can travel either to the Orbitrap - a very accurate and high-resolution mass analyzer that uses the Fourier transform to determine their mass, or to the collision cell, where they can be fragmented. From there, the path leads either back to the Orbitrap or further to the linear ion trap. This serves as the second mass analyzer in the device. It is less accurate and low-resolution, but provides greater sensitivity and the possibility of ion fragmentation. Fragmentation products can be isolated in the trap and fragmented again. This enables analysis in MS³ and even higher levels of fragmentation.</p>
<h5><strong>TSQ Quantiva Triple Quadrupole Mass Spectrometer</strong></h5>
<ul>
<li><a href="https://lcms.labrulez.com/library?instrumentationItems=31-32-35&amp;mainauthorItems=3&amp;search=Quantiva"><strong>Application in the LabRulezLCMS library</strong></a></li>
</ul>
<h5><strong>Leco - Pegasus® 4D GCxGC-TOFMS</strong></h5>
<ul>
<li><a href="https://gcms.labrulez.com/library?instrumentationItems=3-13-2&amp;mainauthorItems=24&amp;search=4D"><strong>Application in the LabRulezGCMS library</strong></a></li>
</ul>
<h4><strong>Liquid chromatography</strong></h4>
<ul>
<li>nano LC Thermo Dionex Ultimate 3000</li>
<li>LC Thermo Dionex Ultimate 3000</li>
</ul>
<h4><strong>Sample preparation</strong></h4>
<ul>
<li>KingFisher Flex</li>
<li>vacuum concentrator</li>
<li>Low flow fraction collector - Spider</li>
<li>Bioruptor pico - sonication bath</li>
</ul>
<h4><strong>Software</strong></h4>
<ul>
<li>Spectronaut 19</li>
<li>FreeStyle</li>
<li>ChromaTOF 4.5</li>
<li>Proteome Discoverer 2.2</li>
<li>Scaffold Q+S 4.6.1</li>
<li>Compound Discoverer 3.3</li>
<li>TraceFinder</li>
</ul>
<h5><strong>Software - Freeware</strong></h5>
<ul>
<li>LipidHunter</li>
<li>MaxQuant</li>
<li>Perseus</li>
<li>Skyline</li>
</ul>
<h3><a href="https://www.biocev.eu/en/services/omics-mass-spectrometry-core-facility.6#core-facility-team"><strong>Team</strong></a></h3>
<h5><strong>Mgr. Karel Harant</strong></h5>
<ul>
<li>Head of Proteomic Core Facilty</li>
</ul>
<h5><strong>Mgr. Pavel Talacko</strong></h5>
<ul>
<li>Non-targeted proteomics, targeted proteomics</li>
</ul>
<h5><strong>RNDr. Petr Žáček, Ph.D.</strong></h5>
<ul>
<li>GC / MS specialist, small molecule analysis</li>
</ul>
<h5><strong>Mgr. Veronika Ševců</strong></h5>
<ul>
<li>researcher, preparation of proteomic samples</li>
</ul>
<h5><strong>RNDr. Petr Žáček, Ph.D.</strong></h5>
<h5><strong>Mgr. Veronika Ševců</strong></h5>
<ul>
<li>researcher, preparation of proteomic samples</li>
</ul>
<h5><strong>Ing. Olga Součková, Ph.D.</strong></h5>
<ul>
<li>LC/MS specialist, small molecule analysis</li>
</ul>


Introducing BIOCEV OMICS Mass Spectrometry Core Facility

<p><strong>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.</strong></p><h5><strong>Handout for whole series</strong></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><span><strong>Part 1: Data Processing for NIST MSMS Search Using Thermo Freestyle Software (My Favorite Approach)</strong></span></h3><p><span>This guide outlines my preferred method for processing data in <strong>Thermo Freestyle software</strong> for <strong>NIST MSMS searches</strong>. These steps streamline data parsing and ensure accurate mass spectral interpretation for <strong>LC-MS and MSMS analyses</strong>.</span></p><h4><span><strong>Equipment &amp; Software</strong></span></h4><ul data-spread="false"><li><span><strong>Thermo Orbitrap MSMS</strong></span></li><li><span><strong>Thermo Freestyle Software</strong></span></li><li><span><strong>NIST 2023 Tandem Library</strong></span></li></ul><h4><span><strong>Orbitrap Data One Acquisition</strong></span></h4><ul data-spread="false"><li>LC-MS and MSMS Analyses</li><li><span>Non-targeted acquisition of MSMS</span></li><li><span>Positive and Negative Mode</span></li><li><span>Photo Detector Array (PDA) and UV-VIS Analyses</span></li><li><span>Example compounds:</span><ul data-spread="false"><li><span><strong>Uvinul 3000</strong></span> <i><span>(MW 214)</span></i></li><li><span><strong>Tinuvin 234</strong></span> <i><span>(MW 447)</span></i></li></ul></li></ul><p><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"></p><h4><span><strong>My Favorite Data Processing Workflow</strong></span></h4><ul><li>shared with me by colleague</li></ul><h5><span><strong>Step 1: Setting Up Freestyle Windows</strong></span></h5><p><span>To optimize the workspace for data processing:</span></p><ol data-spread="false"><li><span>Open <strong>three</strong> windows at the bottom of Freestyle:</span><ul data-spread="false"><li><span><strong>Top window:</strong> Positive ion MS1</span></li><li><span><strong>Second window:</strong> Negative ion MS2</span></li><li><span><strong>Bottom window:</strong> UV or PDA trace</span></li></ul></li><li><span>Save this layout in <strong>Workspace Options/Layouts</strong> to ensure automatic parsing when opening new files.</span></li><li><span>Add the <strong>Nearby Precursors</strong> feature to simplify MSMS selection (except for averaged MS spectra).</span></li></ol><p><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"></p><h5><span><strong>Step 2: Adjusting the Window Layout</strong></span></h5><ol data-spread="false"><li><span>Use <strong>Select Filter</strong> to:</span><ul data-spread="false"><li><span>Change the top window to <strong>positive ion</strong></span></li><li><span>Change the second window to <strong>negative ion</strong></span></li><li><span>Choose <strong>PDA or UV</strong> for the bottom window</span></li></ul></li><li><span>The spectrum window will display the <strong>selected MS/MS data</strong> for further processing.</span></li></ol><p><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"></p><h5><span><strong>Step 3: Processing MSMS Data</strong></span></h5><ol data-spread="false"><li><span>Left-click in the <strong>top box</strong> for the <strong>positive ion chromatogram</strong>.</span></li><li><span>Right-click to <strong>average the spectrum</strong> in the Spectrum box.</span></li><li><span>Subtract background if necessary <strong>before averaging</strong>.</span></li><li><span>Populate the <strong>Avg Range</strong> box and select <strong>Range 1</strong>.</span></li><li><span>Go to <strong>Positive Ion Chromatogram</strong>, specify the range, and confirm.</span></li><li><span>Avoid averaging MS2 spectra when exporting to <strong>NIST search</strong>, as it will convert accurate mass to <strong>nominal mass</strong>.</span></li></ol><p><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"></p><p><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"></p><p><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"></p><h5><span><strong>Step 4: Selecting an MS2 Spectrum for NIST Search</strong></span></h5><ol data-spread="false"><li><span>Identify an <strong>m/z value of interest</strong> (e.g., <strong>m/z 215.0702</strong>).</span></li><li><span>Apply <strong>Chromatogram range filter</strong> to isolate the target ion.</span></li><li><span>The MS2 spectrum will replace the MS1 chromatogram in the top window.</span></li><li><span>Click on the peak of interest to generate an <strong>MS2 spectrum</strong>.</span></li><li><span>Repeat the process for <strong>Negative Ion MS2 (MSMS) spectrum</strong>.</span></li><li><span>Send spectra to <strong>NIST MS search</strong> for <strong>compound identification</strong>.</span></li></ol><p><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"></p><h3><span><strong>Conclusion</strong></span></h3><p><span>Using this workflow, Thermo Freestyle software efficiently processes LC-MS/MS data for <strong>NIST searches</strong>, ensuring accurate identification of unknown compounds. By saving custom layouts and avoiding averaged MS2 spectra, this method maximizes precision and efficiency.</span></p>

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

<h3><strong>Using Artificial Intelligence to Drive MS Workflow Productivity</strong></h3><p><strong>This is the second in a multi-part blog series highlighting the key attributes and impressive benefits of the Stellar mass spectrometer—the first-of-its-kind instrument designed and optimized for highly multiplexed targeted quantitation, ideally suited—for discovery verification. See Part 1 </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5077" aria-label="This is the second in a multi-part blog series highlighting the key attributes and impressive benefits of the Stellar mass spectrometer—the first-of-its-kind instrument designed and optimized for highly multiplexed targeted quantitation, ideally suited—for discovery verification" uw-rm-vague-link-id="https://www.thermofisher.com/blog/analyteguru/why-you-need-a-verification-class-mass-spectrometer/$here" data-uw-rm-vglnk=""><strong>here</strong></a><strong> and stay tuned for more blog posts in the series.</strong><i><strong>&nbsp;</strong></i></p><p>As artificial intelligence (AI) continues to take the modern world by storm, a similar concept in the scientific world called “<strong>automated</strong> <strong>intelligence</strong>” is revolutionizing work for research scientists in their transition from discovery to clinical validation. This version of AI enables you to leverage empirical data, user-defined guidance and instrument performance metrics to manage experiments and dramatically improve productivity.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Why_You_Need_a_Verification_class_Mass_Spectrometer_45a584008f.jpg" alt="Thermo Fisher Scientific: Why you need a verification-class mass spectrometer – Part 2" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Why_You_Need_a_Verification_class_Mass_Spectrometer_45a584008f.jpg 207w," sizes="100vw" width="1024" height="773"></p><p>It’s possible with an all-new instrument—the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149"><strong>Thermo Scientific Stellar mass spectrometer</strong></a> (MS)—which uses AI to transform the process of building methods, data acquisition management and data processing for interpretation and planning next steps.&nbsp; Combined with the unique instrument performance, the Stellar mass spectrometer creates a new paradigm for targeted quantitation.</p><h3 id="h-garbage-in-garbage-out"><strong>Garbage in, garbage out</strong></h3><p>Scientific research is all about data, and just like with any computational work, poor-quality input will always produce faulty or unusable output. This idea is especially true for clinical and translational researchers who perform biomarker verification. The quality of your method development and research will always be determined by several things: <strong>(1)</strong> your existing data (e.g., compound databases, empirical data, in silico predictive models), <strong>(2)</strong> your guidance (e.g., real-time decision-making parameters) and <strong>(3) </strong>the experimental capabilities of your instrument platform.</p><h3 id="h-another-challenge-limitations-in-the-discovery-to-validation-workflow"><strong>Another challenge: limitations in the discovery-to-validation workflow</strong></h3><p>Poor quality input for method development is one challenge, but it’s not the only one. As we discussed in our <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5077">first blog post </a>researchers have long struggled with a technology gap in available instrument platforms. Until recently, there were no single-vendor solutions that offered excellent performance for both discovery and targeted verification. This was a serious limitation that impacted how much researchers could rely on AI for method-building. The old process still required a lot more hands-on optimization, which seriously limited productivity.</p><h3 id="h-finally-automated-intelligence-without-the-drawbacks"><strong>Finally: automated intelligence without the drawbacks</strong></h3><p>With the advent of automated intelligence available on Stellar MS to build targeted methods and manage data acquisition, it’s a whole new ball game. Stellar MS software leverage automated intelligence to boost laboratory productivity, capacity, and lab reputation for high-quality data with faster turnaround times. As a researcher, you can now use automated intelligence to “feed in” your previous work and provide instructions about how to use that information to make decisions and generate new data. Here’s how it works:</p><h3 id="h-how-ai-speeds-method-creation-and-optimization"><strong>How AI speeds method creation and optimization</strong></h3><p>Stellar MS come with the Thermo Scientific software tool <strong>PRM Conductor </strong>in Skyline software for automated method building. It uses automated intelligence to build highly effective, targeted acquisition methods from your discovery data (from either<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?instrumentationItems=33&amp;manufacturerItems=3&amp;search=Thermo+Scientific+Orbitrap"><strong> Thermo Scientific Orbitrap</strong></a>-based MS systems or the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149"><strong>Stellar MS</strong></a>).</p><p>PRM Conductor aids in the generation of targeted-MS2 and MS3 assays. The creation of an assay is broken into three main parts:</p><ol><li>Skyline metadata describing precursor-to-product transitions is used to filter transitions against a set of thresholds. The filters are based on integrated peak area, relative area, signal-to-background, correlation to median transition, LC base peak width, and retention time. Precursors are filtered by whether they have at least a certain number of qualifying transitions, typically three for peptides and two for small molecules.</li><li>Targeted assays are created with a visualization that depicts the concurrency of the assay in terms of how long the instrument would take to acquire data for the precursors as a function of retention time. Given a desired number of acquired points per LC peak and corresponding cycle time, you can see how various experimental parameters affect the feasibility of acquiring data for the qualifying precursors, such as linear ion trap scan rate and acquisition window width.</li><li>Finally, you can decide whether to create multiple assays for all qualifying precursors, or a single assay with a “balanced load” that picks the best N peptides per protein. Instrument methods for the final assay(s) can then be exported, based on a user-defined template method.</li></ol><p>In addition, Stellar MS solutions use <a target="_blank" rel="noreferrer noopener" aria-label="Normalized Collision Energy - open in a new tab" data-uw-rm-ext-link="" uw-rm-external-link-id="https://assets.thermofisher.com/tfs-assets/cmd/warranties/psb104-normalized-collision-energy-technology-en.pdf$normalizedcollisionenergy"><a target="_blank" rel="noopener noreferrer" href="https://assets.thermofisher.com/TFS-Assets/CMD/Warranties/PSB104-Normalized-Collision-Energy-Technology-EN.pdf"><strong>Normalized Collision Energy</strong></a></a><strong> (NCE)</strong> – a well-established principle that automatically compensates for mass dependency by automatically estimating all of the collision energies. NCE bypasses the need for optimization per precursor-to-product ion transition performed by a triple quadrupole mass spectrometer which takes much longer to perform, multiplies the experimental cost due to replicate injections of standards and dedicated research staff time.&nbsp;</p><h3 id="h-how-ai-streamlines-data-acquisition"><strong>How AI streamlines data acquisition</strong></h3><p>With the Stellar MS automated intelligence capabilities, scientists can also speed data acquisition with these tools:</p><p><strong>Dynamic AGC</strong> (automated gain control) helps the system maintain constant ion count and high data quality regardless of ion flux. Essentially, the Stellar MS regulates the number of charges or ions per spectrum by employing a hyper-fast ion gate managing ion accumulation to pass an ideally constant number of ions per spectrum.</p><figure class="image"><img style="aspect-ratio:400/443;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Why_you_need_a_verification_class_mass_spectrometer_Part_2_2db3d7d0df.jpg" alt="Thermo Fisher Scientific: Figure 1: a) Theoretical depiction of how AGC expands the accessible quantitative range of concentrations. b) Assessment of active targets at the start of a cycle, with their minimum acquisition times on the y-axis, and where more intense precursors are white. 3) Expansion of injection times inverse-proportionally to target abundance. " srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Why_you_need_a_verification_class_mass_spectrometer_Part_2_2db3d7d0df.jpg 141w," sizes="100vw" width="400" height="443"></figure><p>As depicted in Figure 1, the ability to vary the injection time expands the dynamic range of quantitation, from a base range given by just the storage and detector saturation limits and multiplies it by the gate linearity range. In the case of the Stellar MS, the pulsed nature of ion trap mass analysis limits the intra-scan dynamic range to about three orders (from 1 to 25k ions in a peak), while the gate can give a linear response from about 3d-6 seconds up to a practical experimental limit, for example 3e-1 seconds.</p><p>Another tool, <strong>Adaptive RT</strong>, dynamically manages PRM acquisition, which means you’ll nearly eliminate the risk of missing data while maximizing target capacity.&nbsp; Missing data requires you to re-run samples, costing time and money. Using the method (generated by PRM Conductor software), you can run one representative sample on the gradient and product ion distribution profiles as a function of retention time are built into the instrument editor, which combines PRM settings and Adaptive RT to manage every microsecond of sample acquisition. The system will continue to update and modify the method to adjust for any unexpected LC perturbation or manage data acquisition across multi-channel UHPLC column formats. For a more in-depth description, check out the <a target="_blank" rel="noreferrer noopener" aria-label="newly published BioRxiv paper - open in a new tab" data-uw-rm-ext-link="" uw-rm-external-link-id="https://www.biorxiv.org/content/10.1101/2024.05.31.596848v1$newlypublishedbiorxivpaper"><a target="_blank" rel="noopener noreferrer" href="https://www.biorxiv.org/content/10.1101/2024.05.31.596848v1">newly published BioRxiv paper</a></a>.</p><h3 id="h-conclusion-automated-intelligence-maximizes-workflow-performance"><strong>Conclusion: Automated intelligence maximizes workflow performance</strong></h3><p>In order to maximize LC-MSn workflow performance for translational verification, you need solutions that streamline all steps while meeting experimental expectations—scale, sensitivity, specificity, throughput, and ease-of-use. Together, automated intelligence on the Stellar MS combined with unique instrument performance can help deliver the ultimate performance to boost your lab’s productivity and prestige.</p><blockquote><p>For more information, explore the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149">Stellar Mass Spectrometer MS</a> or <a target="_blank" rel="noreferrer noopener" aria-label="reach out to your Thermo Fisher Scientific sales representative - open in a new tab" data-uw-rm-ext-link="" uw-rm-external-link-id="https://www.thermofisher.com/us/en/home/global/forms/industrial/stellar-mass-spectrometer-contact.html$reachouttoyourthermofisherscientificsalesrepresentative"><a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/us/en/home/global/forms/industrial/stellar-mass-spectrometer-contact.html">reach out to your Thermo Fisher Scientific sales representative</a></a>.</p><p>Intrigued to learn more about the Stellar MS and how it can improve your work? Watch for more blog posts in this series soon.</p><h5><strong>Visit our&nbsp;</strong><a target="_blank" rel="noreferrer noopener" aria-label="LinkedIn page - open in a new tab" data-uw-rm-ext-link="" uw-rm-external-link-id="https://www.linkedin.com/showcase/chromatography-&amp;-mass-spectrometry/posts/?feedview=all$linkedinpage"><a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/chromatography-&amp;-mass-spectrometry/posts/?feedView=all"><strong>LinkedIn page</strong></a></a><strong>:</strong> #StellarMassSpec #MassSpectrometry #TranslationalResearch</h5><h4><strong>Dr. Scott Peterman</strong></h4><div itemprop="description">Scott Peterman, Ph.D., is Senior Product Marketing Manager for Life Science Mass Spectrometry of the Chromatography and Mass Spectrometry Division of Thermo Fisher Scientific.</div></blockquote>

Why you need a verification-class mass spectrometer – Part 2

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