News from LabRulezLCMS Library - Week 48, 2024

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 25th November 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 you applications by Thermo Fisher Scientific, Shimadzu, Waters Corporation, and Agilent Technologies!

1. Thermo Fisher Scientific: Enhancing detection of hemoglobin variants in clinical research using dried blood spot and high-resolution accurate mass (HRAM) Orbitrap mass spectrometry

• Application

Application benefits

• Direct protein extraction from dried blood spots for protein identification and quantitation
• Identification: utilizing a top-down analysis approach to obtain Hb variant sequences for the characterization of structural mutation
• Quantitation: utilizing intact protein precursor isotopic m/z to determine ratios between target chains and variants
• Minimal sample preparation (<1 hr) in 96 well plate format, easily transferrable to automation using a robotic system
• Confident data processing and review using automated software tools, Thermo Scientific™ ProSightPD™ software, and Thermo Scientific™ TraceFinder™ software

Goal

Develop a top-down approach for hemoglobin variant detection using dried blood spots and a Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer

Introduction

Hemoglobinopathies and thalassemia are the most common genetically determined disorders. They are caused by pathogenic variants in genes that control the production of hemoglobin subunits. Hemoglobin (Hb, approximately 62 kDa) is a tetramer, consisting of two alpha chains and two beta or beta-like chains. It is responsible for physiological oxygen transport in all vertebrates except for the Channichthyidae. Adult Hb consists of 96–98% Hb A (α2β2), 2.0–3.3% Hb A2 (α2δ2), and less than 1.0% Hb F (α2γ2).¹ Hemoglobinopathy is a genetic defect characterized by the abnormal structure of one of the Hb subunits, usually resulting from single amino acid substitution. Thalassemia is characterized by the absence or decreased expression of one of the Hb subunits. The most common hemoglobinopathy is sickle cell disease (SCD, Hb S (β⁶ Glu→Val)). Around 5.2% of the global population carries significant hemoglobinopathy-causing genes with more than 330,000 affected births occurring annually.²

Current techniques used in Hb analysis are electrophoretic and chromatographic assays. However, due to the lack of resolution, they are limited in differentiating isomers (i.e., between Hb C (β⁶ Glu→Lys) and Hb E (β²⁶ Glu→Lys)), very similar masses (i.e., <1 Da difference between normal Hb, Hb C, Hb E, and Hb D-Punjab (β¹²¹ Glu→Gln)), or new variants. Mass spectrometry has gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Targeted protein and peptide quantitation joined this trend by applying various proteomics approaches such as bottom-up, middle-down, and top-down methods. Recently, the top-down approach targeting intact protein biomarkers was enabled by high-resolution accurate-mass (HRAM) mass spectrometers such as Thermo Scientific™ Orbitrap™-based instruments. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using the Orbitrap Exploris 240 mass spectrometer for clinical research.

Conclusion

This study successfully demonstrates the application of an LC-MS/MS top-down approach for detecting hemoglobin variants using dried blood spots and the Orbitrap Exploris 240 mass spectrometer. The method enables direct protein extraction with minimal sample preparation, allowing high-throughput analysis. The high resolution of the Orbitrap Exploris 240 MS effectively isolates and identifies hemoglobin variants, even with minimal mass differences, and detects low-abundance variants such as the Hb delta chain. The ProSightPD software enhances data analysis by generating sequence maps for variant identification. This approach provides a robust and efficient workflow for clinical research, offering significant improvements over traditional methods. This technical note demonstrated the feasibility of hemoglobin variant detection workflow, highlighting its potential for broader clinical research applications.

2. Shimadzu: MRM Based Free Fatty Acids Profiling in Human Plasma and Serum Using LC-MS/MS

• Application

User Benefits

• Simultaneous analysis of 19 free fatty acids in blood was allowed with simple pretreatment.
• The delay column worked to separate the target fatty acids from the undesirable contaminants.
• LCMS-8060RX newly equipped with CoreSpray enables stable and reliable quantitative analysis for trace amount of compounds.

Introduction

Free fatty acids, about 5% of the total fatty acids in the body, are known to be important signal transducers that regulate functions such as insulin secretion. It is also attracting attention as a biomarker for various endocrine disorders¹⁾.

Gas chromatography (GC) or gas chromatography-mass spectrometry (GC/MS) generally requires pretreatment such as methyl esterification for long-chain fatty acids and polyunsaturated fatty acids analysis. Multiple reaction monitoring (MRM) using liquid chromatography-mass spectrometry (LC/MS) enables highly sensitive quantitative analysis of fatty acids without derivatization. However, fatty acids widely found in the environment, such as palmitic acid and stearic acid, are introduced as impurities in LC system. It has been reported that there is difficulty in accurate quantification of these fatty acids in real samples analysis²⁾.

This report presents an example of quantitative analysis of free fatty acids in human plasma and serum using the highly stable LCMS-8060RX system coupled with a delay column.

Conclusion

Simultaneous analysis of 19 free fatty acids in human plasma and serum was performed using a high-performance liquid chromatograph mass spectrometer LCMS-8060RX. Good linearity was obtained in the range of 1-1000 for all compounds, and we also confirmed that the repeatability at lowest calibration point was less than 10%.

The use of a delay column allowed removing the matrix peaks derived from the mobile phase. This analytical method provides more stable and accurate quantitative analysis with simple pretreatment.

3. Agilent Technologies: Determination of Limonin in Citrus Flour by Time of Flight (TOF) LC/MS

• Application

Abstract

In recent years, citrus flour has become a popular additive to meat and dairy plant-based substitutes as it holds water, emulsifies, binds oil, improves texture and stability over time, and is vegan. Citrus flour contains high concentrations of limonin, a limonoid that results in a delayed bitter taste and is undesirable. Industry processes reduce bitter taste in citrus flour by removal or modification of limonin and other limonoids. The method presented here is useful in the quantitation of limonin in foods, food additives, juices, and plants. This method yields good accuracy, precision, linearity, and range in matrix. The use of an Agilent 6230B Time of Flight LC/MS allows limonin to be distinguished and quantitated. Congeners can also be identified and quantitated.

Introduction

The recent rise in popularity of meat and dairy plant-based substitutes have necessitated the use of citrus fiber or flour to replace starches, gums, chemical emulsifiers, and stabilizers. Citrus flour holds water, emulsifies, binds oil, and is vegan. This natural citrus flour improves texture and stability over time and is particularly useful in baked goods, beverages, dairy products, dressings, meats, sauces, frozen foods, pet foods, dairy alternatives, and plant-based meats.

The global citrus flour market size was valued at USD 427.3 million in 2022 and is projected to reach USD 726.9 million by 2032, growing at a compound annual growth rate (CAGR) of 5.7% from 2023 to 2032.¹ The citrus flour market is being driven  in part by the increased focus on health and well-being throughout the world. Being a high-quality dietary fiber source, citrus flour supports satiety and digestive health. Citrus fiber is becoming a sought-after component in formulas with a health focus as consumers become more conscious of the connection between nutrition and overall health and seek goods that have beneficial nutritional qualities and taste good.

Limonin is enriched in citrus fruits and is often found in higher concentrations in seeds, pulp and peel of oranges, grapefruit, lemons, limes, pumellos, bergamots, and mandarins.² Citrus flour is made from leftover dried fibrous material after the fruit is juiced and can have high concentrations of limonin and other limonoids. Limonin and other limonoid compounds contribute to the delayed bitter taste of some citrus food products and is therefore undesirable.

Limonin is a limonoid: a bitter, white, crystalline substance found in citrus and other plants. It is also known as limonoate, D-ring-lactone, and limonoic acid di-delta-lactone.³ Chemically, it is a member of the class of compounds known as furanolactones. Limonin has an exact mass of 470.19406791 g/mol.⁴ The method presented in this application note is useful in the quantitation of limonin in foods, food additives, juices, and plants. The use of a Time of Flight LC/MS allows excellent quantitation of limonin as well as the characterization of other congeners.

Conclusion

The method described in this application note for the determination and quantitation of limonin in citrus flour using an Agilent Time of Flight LC/MS has been shown to be linear, accurate, and precise over typical industrial concentrations. The use of Agilent Time of Flight LC/MS technology also allows the characterization and quantization of congeners and other matrix components. Post-run screening for other compounds is easily accomplished.

4. Waters Corporation: Metabolomics Workflow using a Xevo™ MRT Mass Spectrometer

• Application

Abstract

We demonstrate the acquisition and data processing to identify and pathway profile a typical metabolomic study acquired using a Xevo™ MRT Mass spectrometer coupled with an ACQUITY™ Premier LC System. The resulting data shows a clear unsupervised PCA separation between the three study groups with exogenous or environmental markers being the most significant differences from the different volunteer lifestyles. The Xevo MRT MS delivers rapid, robust data acquisition for large cohort metabolomic studies, capable of providing a high mass resolution of up to 100,000 full width half maxim (FWHM) and consistent sub-ppm mass accuracy, ensuring the analyst has increased confidence in putative feature identification.

Benefits

• Simple and robust workflow delivering excellent data reproducibility and confident results
• A powerful benchtop mass spectrometer routinely providing high mass resolution for both precursor and product ions, enabling separation of compounds with near identical molecular weights e.g. isoforms
• Delivering a consistent mass accuracy <1 ppm RMS, increasing confidence in putative feature identification
• Ability to acquire data at extremely fast acquisition rates, maintaining mass resolution and mass accuracy benefits. Allowing compatibility with shorter chromatographic methods, increasing sample throughput
• Wide dynamic range providing up to 5-orders of linearity, allowing for a single injection to accurately determine the relative concentration of both low and high abundance compounds within a complex matrix

Introduction

Metabolomics is an area of research which aims to identify key metabolic markers originating from phenotypic differences e.g. disease state, environmental changes, treatment regime etc. This could be to improve the detection of disease, to assess impact of pollutants, or to assist with personalized/tailored medical treatment. The metabolome is very complex, containing thousands of analytes with a bias towards smaller molecular weight compounds. Once potential biomarkers have been highlighted using statistical analysis of the data, the challenge can be to confidently identify the compounds of interest.

Higher mass resolution leads to narrower spectral peaks leading to more accurate mass measurement, allowing for tighter mass tolerances for elemental composition and/or database identification searches, improving the confidence in putative identifications. Many small molecules are also observed share similar molecular weights (isoforms). By increasing the mass resolution of the mass spectrometer data clarity is improved, with a higher mass resolution it is possible to separate two compounds with similar molecular weights. With lower mass resolution these isoforms may have merged to form a single spectral peak, leading to erroneous mass determination and incorrect compound identification.

Using a commercially available urine metabolomics study, we demonstrate the benefits of the Xevo MRT QTof mass spectrometer: delivering routine sub-ppm mass accuracy and high mass resolution. This workflow utilizes the software package MARS (MassAnalytica, Barcelona, Spain) for data processing, statistical analysis and putative identification. A more detailed overview of this software and its functionality can be found in the Waters™ application note: Screening Metabolomics of Human urine using a Waters High Resolution QTof Mass Spectrometer and MARS Data Processing Software.¹ The resulting data showed significant changes in exogenous or environmental markers for each of the different volunteer lifestyles.

Conclusion

We demonstrate that the Xevo MRT Mass Spectrometer coupled with an ACQUITY Premier LC System delivers rapid, robust data acquisition for metabolomic studies. When data is imported into MARS software for data processing this platform provides a powerful tool for acquisition, statistical analysis and putative identification of compounds of interest for metabolomic studies.

This workflow provides the acquisition and data processing needed to confidently identify and pathway profile a typical metabolomic study. The resulting data shows a clear unsupervised PCA separation between the three study groups, putatively identifying several exogenous or environmental markers with a sub 1 ppm mass accuracy and fragmentation patterns, which match closely to theoretical. When these analytes are visualized using a trend plot, significant fold changes indicate differentiation between the three smoker levels. The Xevo MRT MS, delivering a high mass resolution of up to 100,000 FWHM and routine sub-ppm mass accuracy, gives the analyst increased confidence in putative feature identification.