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 15th July 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!

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This week we bring to you poster presentations from ASMS 2024 by Agilent Technologies, Shimadzu, Thermo Fisher Scientific, and Waters Corporation!

1. Agilent Technologies: Leveraging the MS1 Dimension and Formula Prediction in Non-Targeted Analysis of PFAS using New FluoroMatch Algorithms: Assessing Confidence and Coverage

• poster - ASMS 2024

FluoroMatch Covers the Entire Data-Processing Workflow, and Supports Various Acquisition Modes

• 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

Conclusions:

• 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.
• 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).

2. Agilent Technologies: Peptide Mapping of Tryptic Digests for mAbs using a novel ECD cell on the 6545XT AdvanceBio LC/Q-TOF Mass Spectrometer

• Poster - ASMS 2024

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

• LC/MS conditions were optimized to detect low-abundant glycopeptide species from NIST and trastuzumab
• MS/MS spectra for glycopeptides using ExD show peptide fragments with intact glycans and excellent sequence coverage along the peptide backbone
• For CID mode, no intact glycan fragments are observed. Instead, HexNAc glycan fragment at m/z 204.086 is observed

3. Shimadzu: Quantitation of free amino acids in human plasma by Single Quadrupole LC-MS

• poster - ASMS 2024

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:

• 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.
• The required sensitivity and chromatographic separation were achieved with the single quadrupole LC-MS.
• The method can quantitatively analyze 37 Amino acids in biological fluids such as plasma in less than 20 minutes.

4. Shimadzu: Benchtop Matrix Assisted Laser Desorption Ionization Mass Spectrometry Imaging of Human Tonsil Proteins using MALDI HiPLEX-IHC Probes

• Poster - ASMS 2024

Introduction:

• 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.
• 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.
• Analysis of a follicular hyperplasia tissue sample showed visible differences from normal human tonsil tissue relating to structural features and cell distributions (Fig. 1)

Conclusion:

• 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.
• The spectra were well-resolved allowing for clear and specific imaging in both normal and clinically significant samples.
• 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.

5. Thermo Fisher Scientific: Confident transformation site localization of PROTAC drug metabolites facilitated by multi-stage fragmentation LC-HRAM-MS

• Poster - ASMS 2024

Abstract:

• Purpose: Confident characterization of PROTAC drug metabolites for transformation site localization and soft-spot analysis.
• 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.
• 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.

Conclusions:

• 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.
• MSn fragmentation data helped to enhance the precision of transformation site localization, particularly for isomeric metabolites and those with high molecular weights.

6. Thermo Fisher Scientific: Unleashing the power of HT-DIA acquisition on Orbitrap Exploris 240 MS – Precise and accurate quantitation at 260 SPD

• Poster - ASMS 2024

Abstract:

Assessing and demonstrating the qualitative and quantitative performance of fast data-independent acquisition (DIA) gradients (<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:

• Excellent quantitation accuracy and precision for small amounts of bacterial and fungal proteomes from mammalian background proteomes
• Increased sample throughput with minimal impact on the quality of the obtained data and proteome coverage
• Outstanding instrument productivity at sample turnover rates up to 250 samples per day
• Interlaboratory reproducibility, long-term stability, and robustness essential for large cohort and multi center studies PO002624

7. Waters Corporation: NON-TARGETED ANALYSIS OF MUSHROOM-CONTAINING COFFEE PRODUCTS USING ION MOBILITY-HIGH RESOLUTION MASS SPECTROMETRY

• Poster - ASMS 2024

INTRODUCTION:

• 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 "functional" mushrooms.
• There is a wide variety of coffee products currently on the market that are blended with extracts from an assortment of different mushroom species.
• In this study, we characterized mushroom coffee products containing up to three mushroom species: chaga, cordyceps, and lion's mane. The chemical fingerprints were compared with that of traditional coffee.

Conclusion:

• 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.
• Trehalose disaccharide displayed variability among the mushroom coffees, but was relatively consistent across the cordyceps, chaga, and lion’s mane capsules.
• 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.

8. Waters Corporation: Ion Mobility Separation Of Marine Natural Products: Characellides from the Deep-sea Sponge Characella pachastrelloides

• Poster - ASMS 2024

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:

• 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
• The added separation power of IMS led to the discovery of two new compounds with the same molecular formula as Characellides C and D.
• 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.