Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 22th July 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!
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<h4>👉 <a href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></h4>
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👉 Need info about different analytical techniques? Peek into <a href="https://gcms.labrulez.com/library">LabRulezGCMS</a> or <a href="https://icpms.labrulez.com/library">LabRulezICPMS</a> libraries.
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This week we bring to you poster presentations from <a href="https://lcms.labrulez.com/events/568">ASMS 2024</a> by Agilent Technologies, Shimadzu, Thermo Fisher Scientific, and Waters Corporation!
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💡 Almost 1000 ASMS posters and presentations <a href="https://lcms.labrulez.com/library?mainauthorItems=284">available in the LabRulezLCMS library</a> (unique search and filtering)
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<h4>1. Waters Corporation: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/2023_asms_king_liver_178c54e990.pdf">HIGH THROUGHPUT PLASMA PROFILING OF HUMAN LIVER DISEASE SAMPLES USING RAPID CHROMATOGRAPHY AND A MULTI- REFLECTING TIME-OF-FLIGHT MASS SPECTROMETER</a></h4>
<ul>
<li>poster - ASMS 2024</li>
</ul>
Introduction: Metabolomics is a valuable research strategy in the determination FIG. 5 FIG. 1 FIG. 7 FIG. 3 of disease progression, disease stratification, the identification of biological markers for early diagnosis and for the development of new therapeutics. Acute-on-chronic liver failure (ACLF) is a condition characterized by systemic inflammation, multiple organ failure, and high short-term mortality1. ACLF develops from the acute decompensation of cirrhosis and currently there is no biomarker identified for the early diagnosis of this disease. Therefore, fully understanding the role metabolites and lipids play in the progression of ACLF and its underlining mechanisms could greatly help determine new diagnostic markers driving development of new therapeutics. Investigating biomarkers for large scale studies requires robust, high-throughput analytical methods.
Rapid microbore metabolic profiling (RAMMP) methods and conventional high-resolution mass spectrometry have previously been shown to considerably reduce analysis time2-3. Here we compare these methods with a vacuum jacketed column technology (VJC)4-5 and ultra high-resolution mass spectrometry6 for the analysis of plasma from liver disease patients by reversed- phase (RP) for lipid and hydrophilic interaction chromatography (HILIC) for small molecules.
Conclusion:
<ul>
<li>Conventional LC-MS methods used for large cohort studies suffer from batch response variation. The rapid chromatographic methods reduce this impact and demonstrated reproducible response across the entire batch of 168 injections per polarity (Fig. 2).</li>
<li>The SELECT SERIES MRT mass spectrometer generated highly accurate mass measurements (&lt;1 ppm), improving annotation confidence for both lipids and small molecules, including essential amino acids and carnitines from the HILIC analysis (Fig. 3).</li>
<li>PCA of the RAMMP data showed good analytical reproducibility from the clustered QC samples and group separation with some overlap between the different disease groups (Fig. 4).</li>
<li>Group comparisons were performed by OPLS-DA where statistically significant features were highlighted for database annotation (Fig. 5).</li>
<li>A number of lipids classes, particularly lysophosphatidylcholines (LPC), phosphatidylcholines (PC) and sphingomyelins (SM), showed dysregulation in the diseased samples when compared to healthy controls and were annotated through Lipostar2 with mass accuracies between 0.1-0.3 ppm (Fig. 6).</li>
<li>For the HILIC small molecule analysis, differences in amino acid response were detected in comparisons between diseased and healthy controls.</li>
<li>The SELECT SERIES MRT MS achieved mass resolution of 170-200K FWHM at 30 Hz compatible with RAMMP (a) and VJC (b) methodologies for example lipids, PC36:1 (0.18 ppm) and SM42:3, O2 (0.16 ppm), with the VJC providing better peak shape and resolution of adjacent peaks (Fig. 7)</li>
<li>Comparison of the RAMMP and VJC negative lipid analysis showed very similar distribution of disease and healthy control groups demonstrating minimal impact on the overall statistics (Fig. 8).</li>
<li>Both RAMMP and VJC methodologies combined with the SELECT SERIES MRT provided highly accurate reproducible metabolic profiling for a biomarker study, with the VJC providing improved peak fidelity complemented by the fast scanning high resolution capabilities of the SELECT SERIES MRT.</li>
<li>This work demonstrated an accurate, reproducible and high throughput metabolomic method for the rapid screening of a large number of biological samples for biomarker discovery.</li>
</ul>
<h4>2. Thermo Fisher Scientific: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_143_characterization_structure_adenopeptin_lc_hram_ms_po143_asms2024_en_7ae893f36c.pdf">In-depth characterization and structure elucidation of non-ribosomal peptides related to adenopeptin using LC-HRAM-MSⁿ</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<h5>Abstract</h5>
Purpose: Characterization of adenopeptin and related peptaibol analogs in a crude fermentation broth extract using LC-HRAM-MSn to identify adenopeptin congeners and elucidate their structures. Methods: Adenopeptin and its analogs were separated using reversed phase chromatography and analyzed using multi-stage fragmentation on a Thermo Scientific Orbitrap IQ-X Tribrid mass spectrometer. 
Results: Several analogs of adenopeptin differing by one or more methyl groups could be detected and the site of their modification could be narrowed to specific amino acid residues based on their fragmentation patterns.
<h5>Conclusions</h5>
The LC/MS analysis of the crude adenopeptin extract revealed the presence of eight analogs above 0.5% relative MS-abundance, mainly resulting from ”demethylations”, i.e., substitution of Aib for Iva residues. Their modification sites could be determined based on shifts in the fragmentation pattern relative to adenopeptin. Additionally, a potentially crucial substitution of pipecolic acid with proline could be detected and verified based on MS3 spectral evidence for compound 4.
<h4>3. Thermo Fisher Scientific: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_146_evaluation_automated_low_flow_lc_ms_setup_po146_asms2024_en_1d935eb142.pdf">Evaluation of an Automated “Quick” Optimization Routine to Streamline Low-Flow LC-MS Setup</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<h5>Abstract</h5>
Purpose: When setting up low flow LC-MS experiments, a user regularly positions an emitter at an arbitrary fixed distance from the inlet based on visual alignment. A prototype ion source featuring an emitter mounted on a 3D motorized stage was used to evaluate a “quick” optimization strategy used to define the active position for subsequent LC-MS measurements.
Methods: The basis of the quick optimization routine was established using m/z- and position-dependent intensity distributions. The routine was evaluated against two methods to manually set the emitter position.
Results: All optimization strategies yielded a statistically equivalent number of protein and peptide identifications. However, the quick optimization routine yielded superior peak area reproducibility that translated into significantly better emitter-to-emitter consistency.
<h4>4. Shimadzu: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/ASMS_poster_PESI_v_DMS_170524_fed75d0c1e.pdf">DPiMS-MS (PESI) combined with vacuum differential mobility spectrometry for rapid forensic analysis.</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Introduction
<ul>
<li>Ambient ionisation (AI) mass spectrometry techniques facilitate rapid turn-around of samples, often in a walk-up format with limited sample preparation, with obvious benefits for forensic applications.</li>
<li>The absence of a chromatographic separation enables sub-minute data acquisition. However, this comes with the cost of greater chance of problems with chemical noise and/or interference between isomeric/isobaric compounds.</li>
<li>One way to remove chemical noise and to separate interfering compounds is to add an ion mobility separation to the method.</li>
<li>Here we implement a novel low pressure differential mobility on a DPiMS-8060 (PESI) system and demonstrate performance for steroids and small molecule drugs in saliva.</li>
</ul>
Conclusion
<ul>
<li>These results demonstrate the potential of vDMS to enhance AIMS by removing chemical noise and separating isobaric species.</li>
<li>DPiMS-vDMS-8060 (PESI) retains the high acquisition speed of PESI while adding an additional dimension of separation.</li>
<li>The additional selectivity may lower the LOQ and reduce false positives.</li>
<li>vDMS-MS methods can be applied in a wide range of complex matrices (e.g. urine, plasma).</li>
</ul>
<h4>5. Shimadzu: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/MP_045_Targeted_quantitative_screening_pesticides_using_high_resolution_DIA_spectral_library_matching_238a221a67.pdf">Targeted quantitative screening pesticides in food matrices using high resolution DIA spectral library matching.</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Overview
<ul>
<li>A single, generic untargeted data independent acquisition (DIA) method was developed for the analysis of extended panels of pesticides in food matrices.</li>
<li>The high-resolution LC-MS/MS method was designed for multi-residue screening and quantitation of pesticides to meet the European Union SANTE 11312/2021 v2 guidelines capable of working with extended target pesticide panels.</li>
<li>The MS and DIA-MS/MS method was applied to quantify a routine panel of pesticides but also included an additional target panel of fungicides for qualitative screening.</li>
</ul>
Conclusions
<ul>
<li>Using a single generic LC-MS and DIA-MS/MS method targeted quantitation using ion ratio confirmation (to meet the EU SANTE guidelines) and extended search capabilities with library identification were supported.</li>
<li>The method results in the quantitation of &gt;250 pesticides at or below the default MRL concentration (10 µg/kg).</li>
</ul>
<h4>6. Shimadzu: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/MP_045_Targeted_quantitative_screening_pesticides_using_high_resolution_DIA_spectral_library_matching_238a221a67.pdf">Identification of positional isomers of linoleic acid containing phospholipids involved in pancreatic ductal adenocarcinoma.</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Overview
<ul>
<li>To enhance lipid identification, Oxygen Attachment Dissociation (OAD) MS/MS has been applied to provide double bond specific dissociation, enabling C=C position assignments.</li>
<li>High resolution OAD MS/MS was applied in positive and negative ion mode to provide further confidence in lipid identification in a pancreatic adenocarcinoma biomarker study</li>
</ul>
Conclusions
<ul>
<li>OAD-MS/MS is a high specificity fragmentation technique to enable C=C positional assignments in lipids. Analysis is easy to perform alongside CID-MS/MS and spectra are easy to interpret.</li>
<li>OAD-MS/MS has been applied to structurally characterize lipids found to be potential markers of PDAC in a previous study using high resolution metabolomics analysis by LC-CID-MS/MS and DPiMSᵗᵐ.</li>
<li>The use of this new technology aims to enhance pancreatic cancer research by providing more comprehensive identification needed to understand the biological roles of the lipids identified as potential markers of PDAC.</li>
</ul>
<h4>7. Agilent Technologies: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_2dlc_6545xt_monitoring_oxidation_mabs_asms_2024_wp030_en_agilent_77f2bb18d5.pdf">Monitoring oxidation in recombinant monoclonal antibodies at subunit level through two-dimensional liquid chromatography coupled with mass spectrometry</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Introduction: Oxidation, induced by reactive oxygen species, is a prevalent chemical modification in monoclonal antibodies (mAbs), affecting amino acid residues such as cysteine (C), methionine (M), tryptophan (W), tyrosine (Y), lysine (K), and others (Gupta et al., 2022). Each residue&#39;s oxidation uniquely influences the protein&#39;s structure and function. Methionine is highly prone to oxidation in mAbs, typically resulting in the formation of a methionine sulfoxide product with increased polarity. These methionine residues are found in both the antigen-binding (Fab) and effector activity (Fc) domains of a mAb, with those situated on the surface being particularly susceptible to oxidation (Hageman et al., 2019). The increasing popularity of two-dimensional liquid chromatography (2D-LC) has led to a significant rise in the integration of two complementary chromatographic techniques, coupled with mass spectrometry (MS), aiming to provide comprehensive insights into mAb post-translational modifications (PTMs) (Sarin et al., 2022). The proposed 2D WCX-RP-MS workflow enables the analysis of mAbs at the subunit level, providing an automated analytical platform for characterizing and identifying oxidized regions within both the Fc and Fab domains.
Conclusions: The oxidized peaks not resolved by 1D methods are successfully resolved by the D2 RPC column with additional HC-LC information. It should be highlighted that the proposed method completely uses MS compatible buffers and is a faster method to identify mAb modification and their correlation in different mAb fragments compared to the tedious bottomup approach. A general mAb bottom-up analysis takes up to 24-48 hours with the risk of sample loss, degradation, and induced modifications. In the present study, sample preparation took only 4-10 hours (depending on oxidation incubation), and sample analysis took only 3 hours. Hence, 7-13 hours is enough for the subunit analysis with a low risk of sample loss and degradation. The present study focuses on oxidation as the chemical modification of interest, but the workflow can be extended to other critical charge modifications easily resolved by the WCX column.
<h4>8. Agilent Technologies: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/po_2dlc_6545xt_monitoring_oxidation_mabs_asms_2024_wp030_en_agilent_77f2bb18d5.pdf">Flash Characterization of mAbs Using a Combination of Reagents via Automated On-Line Microdroplet Reaction</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Introduction: Recently, an unmodified Agilent Jet Stream ESI source (AJS) was used to demonstrate “Flash Characterization” of mAbs by microdroplet facilitated reductions or enzyme digestions with good reproducibility and high yield 1. These results motivated us to evaluate treatments that combined enzymatic digestion, reduction, and/or deglycosylation of mAb in a single microdroplet experiment. New optimization parameters for the Agilent Injection Program on Agilent 1290 Infinity II LC were presented. Microdroplet experiments, including GlycINATOR ® deglycosylation, the combination of IdeS cleavage and DTT reduction and IdeS cleavage and GlycINATOR ® deglycosylation were examined for reproducibility, intact mAb remaining after treatments and simplified subunit characterization. GlycINATOR is an endoglycosidase that cleaves N-linked glycans and leaves the core GlcNAc residue with or without a fucose on the asparagine. Additional antibodies of Herceptin and sigmamab are also studied with the combination treatment in the microdroplet reaction.
Conclusions: 
<ul>
<li>Flash Characterization of mAbs with an automatic injector, combination treatments, and microdroplet reaction in the AJS source demonstrates simplification, reproducibility, and cost reduction of protein characterization. </li>
<li>Combination treatments are viable with microdroplet reactions to accelerate the reduction, cleavage, and deglycosylation of native IgG1 significantly.</li>
</ul>

News from LabRulezLCMS Library - Week 30, 2024

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)

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 4: Data Processing for NIST MSMS Search using Thermo Freestyle Software (NIST Search Type Demonstration)</h3><h4>Equipment &amp; Software</h4><ul><li>Thermo Orbitrap MSMS</li><li>Thermo Freestyle Software</li><li>NIST 2023 Tandem Library</li></ul><h4>Streamlining Data Processing for Mass Spectrometry</h4>Mass spectrometry is a powerful tool for compound identification, but its efficiency depends largely on how data is processed and analyzed. By utilizing Thermo Freestyle Software in combination with NIST MSMS Search, researchers can enhance the accuracy and speed of their spectral identifications.<h4>NIST MSMS Search v3.0 – Simplified and Enhanced</h4>The NIST MSMS Search version 3.0 introduces an improved interface, making search settings more intuitive. This version allows for:<ul><li>Spectrum import flexibility – Multiple ways to import and process spectra</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Typical_Settings_for_NIST_MS_Search_3_0_Tandem_Mode_dc120f16ec.jpg" alt="James Little: Typical Settings for NIST MS Search 3.0 (Tandem Mode)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Typical_Settings_for_NIST_MS_Search_3_0_Tandem_Mode_dc120f16ec.jpg 245w," sizes="100vw" width="1574" height="664"><ul><li>Various search modes<ul><li>Identity MS/MS: searches spectra restricting by the precursor ion and its specified m/z</li><li>Identity/High Resolution: No Precursor, just compares the overall fit of unknown spectra to reference spectra</li><li>Similarity/Hybrid MS/MS - very useful if a related compound is present in the reference libraries, but the actual compound is not present</li></ul></li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Library_Search_Options_for_NIST_MS_Search_3_0_Tandem_Mode_bb8ca30d1e.jpg" alt="James Little: Library Search Options for NIST MS Search 3.0 (Tandem Mode)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Library_Search_Options_for_NIST_MS_Search_3_0_Tandem_Mode_bb8ca30d1e.jpg 245w," sizes="100vw" width="1569" height="786"><ul><li>Advanced filtering options – Remove low-intensity ions and electronic noise</li><li>Customizable settings – Users can save configurations for future searches</li></ul><h4>Configuring Search Settings for Optimal Performance</h4>To get the most accurate results, users should:<ul><li>Select the appropriate MSMS libraries – NIST 2023 includes essential databases.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Select_the_appropriate_MSMS_libraries_42e991834a.jpg" alt="James Little: Select the appropriate MSMS libraries" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Select_the_appropriate_MSMS_libraries_42e991834a.jpg 199w," sizes="100vw" width="1036" height="814"><ul><li>Adjust precursor m/z tolerance – Ensure compatibility with your mass spectrometer.</li><li>Use the “Best Matching” option – Simplifies results by filtering only the best spectrum using the CAS Number as a filter</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Other_Settings_for_NIST_MS_Search_3_0_Tandem_Mode_3b222cdcf4.jpg" alt="James Little: Other Settings for NIST MS Search 3.0 (Tandem Mode)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Other_Settings_for_NIST_MS_Search_3_0_Tandem_Mode_3b222cdcf4.jpg 245w," sizes="100vw" width="1575" height="869"><ul><li>Save search configurations – Avoid resetting parameters for each new session.</li></ul>⚠️ Important! Save your settings for future searches using the Save Configuration option, and restore them when needed to maintain consistency in your analyses.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Important_Save_Your_Settings_for_Future_Sessions_f614f0a27e.jpg" alt="James Little: Important! Save Your Settings for Future Sessions." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Important_Save_Your_Settings_for_Future_Sessions_f614f0a27e.jpg 245w," sizes="100vw" width="1072" height="632"><h4>Conclusion</h4>Utilizing NIST MSMS Search v3.0 within Thermo Freestyle Software allows for a faster, more accurate, and customizable approach to compound identification. By selecting the appropriate search type and optimizing settings, researchers can enhance the reliability of their mass spectrometry workflows.

Data Processing Using Thermo Freestyle Software with NIST MSMS Search for Compound Identifications (NIST Search Type Demonstration)

Quantification of Gd species in a tetrameric Gd-based contrast agent using HPLC-ICP-MS

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/article/4978">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/article/5160">Different NIST MSMS Searches Demonstrated</a></li><li>Part 5: <a target="_blank" rel="noopener noreferrer" href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/Part_5_Elemental_Composition_demonstration_27e151914b.pdf">Elemental Processing within Thermo Freestyle</a></li></ul><h3>Part 1A: Data Processing Using Thermo Freestyle Software with NIST MSMS Search for Compound Identifications (“Nearby Precursors” Enhancement)</h3><h4>Equipment &amp; Software</h4><ul><li>Thermo Orbitrap MSMS</li><li>Thermo Freestyle Software</li><li>NIST MSMS Search</li></ul>Mass spectrometry data processing is a critical step in analyzing complex chemical mixtures. This article focuses on a preferred method for processing data using Thermo Freestyle software, with an added enhancement—Nearby Precursors. This approach allows for efficient spectral analysis and identification of compounds, improving workflow efficiency and accuracy.<h3>Step-by-Step Guide to Data Processing</h3><h4>1. Setting Up the Workspace</h4>To streamline data analysis, follow these steps to configure the workspace in Thermo Freestyle:<ul style="list-style-type:disc;"><li style="tab-stops:list 36.0pt;">Open three windows at the bottom of Freestyle:&nbsp;<ul style="list-style-type:circle;"><li style="tab-stops:list 72.0pt;">Top Window: Positive ion MS1</li><li style="tab-stops:list 72.0pt;">Middle Window: Negative ion MS2</li><li style="tab-stops:list 72.0pt;">Bottom Window: UV or PDA trace</li></ul></li><li style="tab-stops:list 36.0pt;">Save the layout using Workspace Options/Layouts to ensure automatic application when new files are opened.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Part_1_A_Setting_Up_the_Workspace_e21f14fc45.jpg" alt="James Little: Part 1A - Setting Up the Workspace" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Part_1_A_Setting_Up_the_Workspace_e21f14fc45.jpg 245w," sizes="100vw" width="1558" height="386"><h4>2. Selecting Ranges and Filters</h4><ul style="list-style-type:disc;"><li style="tab-stops:list 36.0pt;">Keep adding windows until you have three total.</li><li style="tab-stops:list 36.0pt;">Use the filter function to configure:&nbsp;<ul style="list-style-type:circle;"><li style="tab-stops:list 72.0pt;">Positive ion in the top window</li><li style="tab-stops:list 72.0pt;">Negative ion in the second window</li><li style="tab-stops:list 72.0pt;">UV/PDA trace in the bottom window</li></ul></li><li style="tab-stops:list 36.0pt;">The positive and negative ion options are the last two items in the filter list.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Part_1_A_Selecting_Ranges_and_Filters_34d956ade6.jpg" alt="James Little: Part 1A - Selecting Ranges and Filters" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Part_1_A_Selecting_Ranges_and_Filters_34d956ade6.jpg 245w," sizes="100vw" width="1486" height="790"><h4>3. Spectrum Window and Data Processing</h4>Once the workspace is set up, it provides three working areas:<ul style="list-style-type:disc;"><li style="tab-stops:list 36.0pt;">Processing boxes to refine data selection</li><li style="tab-stops:list 36.0pt;">Function selection boxes to choose necessary analyses</li><li style="tab-stops:list 36.0pt;">Spectrum visualization for MS and MSMS data</li></ul>Tip: Always save the layout so it automatically loads when opening new files.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Part_1_A_Spectrum_Window_and_Data_Processing_d0b532ee4d.jpg" alt="James Little: Part 1A - Spectrum Window and Data Processing" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Part_1_A_Spectrum_Window_and_Data_Processing_d0b532ee4d.jpg 245w," sizes="100vw" width="1500" height="859"><h3>Enhancing Analysis with Nearby Precursors</h3>The Nearby Precursors feature allows users to quickly access and analyze MS2 spectra from associated peaks. Here’s how to use it effectively:<h4>1. Selecting Peaks and Generating MS2 Spectra</h4><ul style="list-style-type:disc;"><li style="tab-stops:list 36.0pt;">Left-click on a peak in either the positive or negative chromatogram (e.g., peak at 10.05 minutes).</li><li style="tab-stops:list 36.0pt;">The corresponding MS2 spectrum will appear in the second window.</li><li style="tab-stops:list 36.0pt;">Double left-click on the purple circle associated with the ion to display the MSMS spectrum.</li></ul>Note: Nearby Precursors cannot be used with an averaged MS spectrum!<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Part_1_A_Nearby_Precursors_Option_2435bc6a66.jpg" alt="James Little: Part 1A - Nearby Precursors Option" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Part_1_A_Nearby_Precursors_Option_2435bc6a66.jpg 245w," sizes="100vw" width="1169" height="633"><h4>2. Sending Spectra to NIST MSMS Search</h4>Once the MS2 spectrum is displayed:<ul style="list-style-type:disc;"><li style="tab-stops:list 36.0pt;">Select the bar above the spectrum with a left-click.</li><li style="tab-stops:list 36.0pt;">Send the spectrum to NIST search for further identification.</li><li style="tab-stops:list 36.0pt;">Remove unwanted spectra by clicking the ‘x’ in the box.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Part_1_A_Nearby_Precursors_Option_NIST_MSMS_74e962efcd.jpg" alt="James Little: Part 1A - Nearby Precursors Option - NIST MSMS" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Part_1_A_Nearby_Precursors_Option_NIST_MSMS_74e962efcd.jpg 245w," sizes="100vw" width="1477" height="673"><h4>Troubleshooting: Recovering Deleted Spectra Windows</h4>If all MS and MS2 spectrum windows are accidentally removed:<ol><li style="tab-stops:list 36.0pt;">Navigate to Workspace Options &gt; Spectrum &gt; Insert View.</li><li style="tab-stops:list 36.0pt;">Reinsert the necessary spectrum windows to continue the analysis.</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/James_Little_Part_1_A_If_You_Delete_All_the_MS_and_MS_2_Windows_by_Mistake_b62af649db.jpg" alt="James Little: Part 1A - If You Delete All the MS and MS2 Windows by Mistake" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_James_Little_Part_1_A_If_You_Delete_All_the_MS_and_MS_2_Windows_by_Mistake_b62af649db.jpg 205w," sizes="100vw" width="998" height="758"><h4>Conclusion</h4>The Preferred Approach combined with the Nearby Precursors enhancement provides a streamlined and efficient way to process LC-MS and MSMS data using Thermo Freestyle software. This method ensures better spectral organization, easier peak selection, and faster searches in NIST MSMS databases.

Data Processing Using Thermo Freestyle Software with NIST MSMS Search for Compound Identifications (“Nearby Precursors” Enhancement)

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