Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 15th July 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!
<blockquote>
<h4>👉 <a href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></h4>
</blockquote>
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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!
<h4>1. Agilent Technologies: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_fluoromatch_asms_2024_tp113_en_agilent_93ee360315.pdf">Leveraging the MS1 Dimension and Formula Prediction in Non-Targeted Analysis of PFAS using New FluoroMatch Algorithms: Assessing Confidence and Coverage</a></h4>
<ul>
<li>poster - ASMS 2024</li>
</ul>
FluoroMatch Covers the Entire Data-Processing Workflow, and Supports Various Acquisition Modes
<ul>
<li>Data was acquired on an Agilent 6546 LC/Q-TOF for this study to assess PFAS in dried blood spot cards (paper) and blood on dried blood spot cards</li>
</ul>
Conclusions:
<ul>
<li>FluoroMatch can be used to rapidly annotate PFAS in an automatic fashion, determine unknowns and expand annotation using an interactive visualizer. Formula prediction provides a significant benefit by providing evidence for annotation for molecules which are considered tentative using other lines of evidence, or for which the signal is too low to get quality MS/MS.</li>
<li>The FluoroMatch formula prediction algorithm is highly sophisticated and has several new or optimized approaches for formula prediction of fluorine containing compounds. With the additional use of homologous series assigning the predicted formula with the most common motifs for all members of a series, the resulting annotations are highly confident (0% false positives in this case for the four series with confident assignments).</li>
</ul>
<h4>2. Agilent Technologies: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_exd_6545xt_mabs_glycopeptide_asms_2024_mp665_en_agilent_01a5c15cdd.pdf">Peptide Mapping of Tryptic Digests for mAbs using a novel ECD cell on the 6545XT AdvanceBio LC/Q-TOF Mass Spectrometer</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Introduction:
Post-translation modifications (PTMs) on monoclonal antibodies (mAb) play an important role in the safety, efficacy, and binding of a therapeutic to its target [1]. Common examples of PTMs that seek to be identified include glycosylation and phosphorylation. One challenge in the identification of these PTMs is that dissociation techniques such as collision-induced dissociation (CID) can break apart these fragile modifications. Here, we describe the use of an electron-based dissociation technique (ExD) that supplies low-energy electrons for the fragmentation of tryptic digests that contain glycosylation at asparagine consensus sites (NXS/T). The fragmentation of trastuzumab tryptic digest on a 6545XT AdvanceBio LC/Q-TOF using CID is compared to illustrate that CID alone will fragment these labile PTMs.
Conclusions: The Agilent 6545XT AdvanceBio LC/Q-TOF with ExD cell enables glycopeptide analysis with excellent sequence coverage assessed using MassHunter BioConfirm 12.1
<ul>
<li>LC/MS conditions were optimized to detect low-abundant glycopeptide species from NIST and trastuzumab</li>
<li>MS/MS spectra for glycopeptides using ExD show peptide fragments with intact glycans and excellent sequence coverage along the peptide backbone</li>
<li>For CID mode, no intact glycan fragments are observed. Instead, HexNAc glycan fragment at m/z 204.086 is observed</li>
</ul>
<h4>3. Shimadzu: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_2024_Th_P_178_e34df60086.pdf">Quantitation of free amino acids in human plasma by Single Quadrupole LC-MS</a></h4>
<ul>
<li>poster - ASMS 2024</li>
</ul>
Introduction: Amino acids are organic compounds that combine to form proteins. They are typically known as the building blocks of protein. Quantitative analysis of amino acids from biological fluids is of great importance in the clinical diagnosis of inborn errors of amino acid metabolism. The assessment of amino acids in body fluids is needed for the diagnosis and treatment of metabolic diseases. We have here performed a high-throughput method for analysis of amino acids in plasma samples. The method combines classical protein hydrolysis and derivatization with fast separation by UHPLC and detection by a single quadrupole mass spectrometer
Conclusion:
<ul>
<li>Liquid chromatography triple quadrupole mass spectrometry (LCMS/MS) analysis of amino acids is becoming the method of choice by more and more laboratories because of its speed, sensitivity, and increased specificity. Although LC-MS/MS instruments are more selective and sensitive than LC-MS single quadrupole instruments, LC-MS can also be used in the quantitative analysis of amino acids when the necessary conditions are provided.</li>
<li>The required sensitivity and chromatographic separation were achieved with the single quadrupole LC-MS.</li>
<li>The method can quantitatively analyze 37 Amino acids in biological fluids such as plasma in less than 20 minutes.</li>
</ul>
<h4>4. Shimadzu: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_2024_TP_198_Shimadzu_MALDI_IHC_Benchtop_bad9f3fdc3.pdf">Benchtop Matrix Assisted Laser Desorption Ionization Mass Spectrometry Imaging of Human Tonsil Proteins using MALDI HiPLEX-IHC Probes</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Introduction:
<ul>
<li>MALDI mass spectrometry imaging (MSI) is a versatile technique capable of characterising lipid, polysaccharide, metabolite, pharmaceutical, protein and peptide distributions within a wide variety of samples. Recently, MSI has been combined with commercially available antibody probes containing photocleavable mass-tags (PC-MTs) which target specific proteins in what has been termed MALDIimmunohistochemistry (MALDI-IHC) MSI.</li>
<li>In this study, we show imaging of FFPE human tonsil sections on an entry level benchtop MALDI-8020 using both a multiplex of 6 antibodies and a cancer marker screening panel consisting of a multiplex of 20 antibodies at a stage step size of 30μm, demonstrating a reliable, lowcost approach with good quality results.</li>
<li>Analysis of a follicular hyperplasia tissue sample showed visible differences from normal human tonsil tissue relating to structural features and cell distributions (Fig. 1)</li>
</ul>
Conclusion:
<ul>
<li>We have successfully mapped up to 20 biomarkers with a stage step size of 30µm using a low cost, linear benchtop MALDI-TOF mass spectrometer in conjunction with the Miralys probe kit.</li>
<li>The spectra were well-resolved allowing for clear and specific imaging in both normal and clinically significant samples.</li>
<li>The Shimadzu MALDI-8020 and MALDI-8030 MALDI-TOF mass spectrometers are robust systems with class leading sensitivity and resolution capable of a wide variety of applications including, as demonstrated here, MALDI imaging. These systems can provide an invaluable contribution to any research laboratory.</li>
</ul>
<h4>5. Thermo Fisher Scientific: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_144_transformation_protac_metabolites_multi_stage_lc_hram_ms_po144_asms2024_en_4db8598a6b.pdf">Confident transformation site localization of PROTAC drug metabolites facilitated by multi-stage fragmentation LC-HRAM-MS</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<h5>Abstract:</h5>
<ul>
<li>Purpose: Confident characterization of PROTAC drug metabolites for transformation site localization and soft-spot analysis.</li>
<li>Methods: Drug metabolites were analyzed using MSn fragmentation data acquisition on a Thermo Scientific Orbitrap Ascend Biopharma Tribrid mass spectrometer and data interpretation in Thermo Scientific Compound Discoverer 3.3 SP3 software.</li>
<li>Results: PROTAC drug metabolism was found to be extensive for the investigated compounds. Among the detected metabolites, several isomeric compounds were found where MSn fragmentation data could be used to reduce ambiguity in the transformation site localization, especially for metabolites with higher molecular weights.</li>
</ul>
Conclusions:
<ul>
<li>The utility of flexible MS data acquisition methods for metabolite identification could be demonstrated with two representative PROTAC compounds, MZ1 and dBET1, which exhibit extensive metabolism in vitro.</li>
<li>MSn fragmentation data helped to enhance the precision of transformation site localization, particularly for isomeric metabolites and those with high molecular weights.</li>
</ul>
<h4>6. Thermo Fisher Scientific: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_145_unleashing_power_ht_dia_orbitrap_exploris_240_ms_accurate_quantitation_po145_asms2024_en_2a2f215fdf.pdf">Unleashing the power of HT-DIA acquisition on Orbitrap Exploris 240 MS – Precise and accurate quantitation at 260 SPD</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
<h5>Abstract:</h5>
Assessing and demonstrating the qualitative and quantitative performance of fast data-independent acquisition (DIA) gradients (&lt;270SPD) on a Thermo Scientific Orbitrap Exploris 240 mass spectrometer (MS), Thermo Scientific Vanquish Neo UHPLC system and micropillar array-based 5.5 cm Thermo Scientific µPAC Neo high-throughput HPLC column.
Conclusions: The performance of a novel high throughput (HT) DIA workflow for label-free quantitation was demonstrated by combining an Orbitrap Exploris 240 mass spectrometer with fast capillary flow chromatography on a high throughput µPAC Neo HPLC column and Vanquish Neo UHPLC system. Key performance criteria of the setup/workflow include:
<ul>
<li>Excellent quantitation accuracy and precision for small amounts of bacterial and fungal proteomes from mammalian background proteomes</li>
<li>Increased sample throughput with minimal impact on the quality of the obtained data and proteome coverage </li>
<li>Outstanding instrument productivity at sample turnover rates up to 250 samples per day </li>
<li>Interlaboratory reproducibility, long-term stability, and robustness essential for large cohort and multi center studies PO002624</li>
</ul>
<h4>7. Waters Corporation: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/2024_asms_hatch_mushroom_coffee_9c919673f3.pdf">NON-TARGETED ANALYSIS OF MUSHROOM-CONTAINING COFFEE PRODUCTS USING ION MOBILITY-HIGH RESOLUTION MASS SPECTROMETRY</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
INTRODUCTION:
<ul>
<li>In recent years, the health benefits of mushrooms—including anti- inflammatory effects1 and improved cognitive function2—have led to huge growth in the number of food and supplement products containing so- called &quot;functional&quot; mushrooms.</li>
<li>There is a wide variety of coffee products currently on the market that are blended with extracts from an assortment of different mushroom species.</li>
<li>In this study, we characterized mushroom coffee products containing up to three mushroom species: chaga, cordyceps, and lion&#39;s mane. The chemical fingerprints were compared with that of traditional coffee.</li>
</ul>
Conclusion:
<ul>
<li>A series of oligosaccharides were observed in mushroom coffees and cordyceps capsules, but not chaga or lion’s mane capsules, nor in traditional coffee. These oligosaccharides may contribute to enhanced health benefits of mushroom coffee.</li>
<li>Trehalose disaccharide displayed variability among the mushroom coffees, but was relatively consistent across the cordyceps, chaga, and lion’s mane capsules.</li>
<li>Taken together, these observations suggest that the extraction process (and/or the parts of the mushrooms extracted) governs the oligosaccharide content more than the specific mushroom species included in a product, though further testing is needed.</li>
</ul>
<h4>8. Waters Corporation: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/2024_asms_sarah_characellides_828ec17fb4.pdf">Ion Mobility Separation Of Marine Natural Products: Characellides from the Deep-sea Sponge Characella pachastrelloides</a></h4>
<ul>
<li>Poster - ASMS 2024</li>
</ul>
Introduction: Characella pachastrelloides was first described in 1876 as part of a collection of deep- sea sponges.1 Deep-sea sponges are a source of unique natural products partly due to the extreme environment they inhabit. This study looks at a group of glycolipopeptides known as characellides that were isolated from C. pachastrelloides and found to be bioactive metabolites.2,3 While HRMS can be used for some structural characterization, full elucidation of natural product structures remains challenging. The ‘gold-standard’ method for structural characterization of natural product compounds is nuclear magnetic resonance (NMR). However, certain compounds can be difficult to isolate or at concentrations too low for quality NMR analysis. This study investigates if ion mobility (IMS) can be another tool for structural assignments of these compounds.
Conclusions:
<ul>
<li>The structurally similar characellide isomers A and B were able to be separated in ion mobility space by utilizing multiple passes in the Cyclic IMS device</li>
<li>The added separation power of IMS led to the discovery of two new compounds with the same molecular formula as Characellides C and D.</li>
<li>Additional related compounds were identified with molecular networking and their CCS values were calculated to give insights into the structural diversity of this unique set of compounds.</li>
</ul>

News from LabRulezLCMS Library Week 29, 2024

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!
<blockquote>
<h4>👉 <a href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></h4>
</blockquote>
<blockquote>
👉 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.
</blockquote>
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!
<blockquote>
💡 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)
</blockquote>
<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>

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