bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2022‒09‒25
67 papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Methods Mol Biol. 2023 ;2571 189-206
      Metabolites represent the most downstream level of the cellular organization. Hence, an in vitro untargeted metabolomics approach is extremely valuable to deepen the understanding of how endogenous metabolites in cells are altered under a given biological condition. This chapter describes a robust liquid chromatography-high-resolution mass spectrometry-based metabolomics and lipidomics platform applied to cell culture extracts. The analytical workflow includes an optimized sample preparation procedure to cover a wide range of metabolites using liquid-liquid extraction and validated instrumental operation procedures with the implementation of comprehensive quality assurance and quality control measures to ensure high reproducibility. The lipidomics platform is based on reversed-phase liquid chromatography for the separation of slightly polar to apolar metabolites and covers a broad range of lipid classes, while the metabolomics platform makes use of two hydrophilic interaction liquid chromatography methods for the separation of polar metabolites, such as organic acids, amino acids, and sugars. The chapter focuses on the analysis of cultured HepaRG cells that are derived from a human hepatocellular carcinoma; however, the sample preparation and analytical platforms can easily be adapted for other types of cells.
    Keywords:  Cell culture extracts; HepaRG cells; High-resolution mass spectrometry; Hydrophilic interaction liquid chromatography; Quality assurance and quality control; Reversed-phase liquid chromatography; Untargeted lipidomics; Untargeted metabolomics
  2. Methods Mol Biol. 2022 ;2546 411-420
      Plasma and serum are rich sources of proteins that are commonly used for clinical proteome profiling and biomarkers discovery. However, high-throughput plasma proteome profiling and quantitative analysis using mass spectrometry are challenging because of the large dynamic range of protein abundance and complexity. To overcome these challenges, we developed a convenient high-throughput workflow of depleted plasma using the 4D-Proteomics feature of the Bruker timsTOF Pro mass spectrometer with data-dependent (PASEF) and data-independent acquisition (diaPASEF) method that can potentially be used in a clinical proteome profiling and biomarker discoveries. This workflow is robust, optimal for high throughput, high proteome depth, and is reproducible. In our sample preparation steps, we used immuno-depletion steps to remove high-abundance plasma proteins, and without any further cleanup steps, we can use depleted plasma samples directly for enzymatic digestion. Immuno-depletion steps and 4D-Proteomics features of timsTOF Pro increase the plasma proteome depth, and accuracy with the identification of >800 protein groups.
    Keywords:  Biomarkers; High-throughput; Immuno-depletion; PASEF; Plasma; Spectral library; Spectronaut; TimsTofPro; diaPASEF
  3. Methods Mol Biol. 2023 ;2571 83-94
      Capillary electrophoresis-mass spectrometry (CE-MS) is an ideal method for analyzing various metabolites in biological samples. CE-MS can simultaneously identify and quantify hundreds of charged metabolites using only two acquisition methods for positively and negatively charged metabolites. Furthermore, CE-MS is commonly used for analyzing biological samples to understand the pathology of diseases at the metabolic level and biofluid samples, such as blood and urine, to explore biomarkers. Here, we introduce a protocol that delineates the handling of clinical samples to ensure that the CE-MS analysis yields reproducible quantified data. We have focused on sample collection, storage, processing, and measurement. Although the implementation of rigorous standard operating protocols is preferred for enhancing the quality of the samples, various limitations in an actual clinical setting make it difficult to adhere to strict rules. Therefore, the effect of each process on the quantified metabolites needs to be evaluated to design a protocol with acceptable tolerances. Furthermore, quality controls and assessments to handle clinical samples are introduced.
    Keywords:  Biofluid; Capillary electrophoresis–mass spectrometry; Clinical samples
  4. Methods Mol Biol. 2022 ;2546 401-409
      Utilizing biofluids to identify cancer biomarkers has received considerable attention in the past decade. In this regard, serum and urine are convenient biofluids to noninvasively recapitulate information usually indicated by traditional tissue biopsies. In particular, we are interested in exploring the extracellular vesicle (ECV)-containing compartment of these fluids as a targeted source for cancer biomarker discovery. ECVs are membrane-enclosed particles, comprising of various fractions including exosomes, microvesicles, and apoptotic bodies. In both physiological and pathological states such as cancer, ECVs carry a rich load of molecular and protein cargoes, which aid in mediating intercellular communication between cells from various tissue types. Here we successfully enriched ECVs using a simple, low-cost, optimized method that we have developed; it is generalizable for the analysis of ECVs from multiple sample types. Such procedures are necessary as ECVs are nanoparticles that contain a treasure trove of large numbers of biomarkers each present at very low levels. Sample processing procedures can enrich for these vesicles and allow for the enhanced detection of proteins in downstream applications such as comprehensive proteomics methods using data-independent acquisition (DIA) and LC-MS/MS.
    Keywords:  Cancer; Data-independent acquisition; Exosomes; Extracellular vesicles; Liquid biopsy; Proteomics
  5. Biomolecules. 2022 Sep 08. pii: 1265. [Epub ahead of print]12(9):
      Despite the increasing popularity of liquid chromatography-mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated methods for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantification of pure lipids. However, the impact of complex lipid sample complexity and purity on total lipid quantification accuracy has not been investigated. Here, we report comprehensive assessment of the sample matrix on the accuracy of lipid quantification using Attenuated Total Reflectance (ATR)-FTIR and establish a simple workflow for lipidomics sample quantification. We show that both pure and complex lipids show characteristic FTIR vibrations of CH- and C=O-stretching vibrations, with a quantitative range of 40-3000 ng and a limit of detection of 12 ng, but sample extraction method and local baseline subtraction during FTIR spectral processing significantly impact lipid quantification via CH stretching. To facilitate sample quality screening, we developed the Lipid Quality (LiQ) score from a spectral library of common contaminants, using a ratio of peak heights between CH stretching vibrations maxima and the collective vibrations from amide/amine, CH-stretching minima and sugar moieties. Taking all tested parameters together, we propose a rapid FTIR workflow for routine lipidomics sample quality and quantity assessment and tested this workflow by comparing to the total LC-MS intensity of targeted lipidomics of 107 human plasma lipid extracts. Exclusion of poor-quality samples based on LiQ score improved the correlation between FTIR and LC-MS quantification. The uncertainty of absolute quantification by FTIR was estimated using a 795 ng SPLASH LipidoMix standard to be <10%. With low sample requirement, we anticipate this simple and rapid method will enhance lipidomics workflow by enabling accurate total lipid quantification and normalization of lipid quantity for MS analysis.
    Keywords:  FTIR; chemical contaminants; lipids; mass spectrometry; phospholipids; sphingolipids; triglycerides
  6. Methods Mol Biol. 2023 ;2571 241-255
      Mass spectrometry (MS)-based metabolomics provides high-dimensional datasets; that is, the data include various metabolite features. Data analysis begins by converting the raw data obtained from the MS to produce a data matrix (metabolite × concentrations). This is followed by several steps, such as peak integration, alignment of multiple data, metabolite identification, and calculation of metabolite concentrations. Each step yields the analytical results and the accompanying information used for the quality assessment of the anterior steps. Thus, the measurement quality can be analyzed through data processing. Here, we introduce a typical data processing procedure and describe a method to utilize the intermediate data as quality control. Subsequently, commonly used data analysis methods for metabolomics data, such as statistical analyses, are also introduced.
    Keywords:  Data processing; Mass spectrometry; Multivariate analysis; Statistical analysis
  7. Methods Mol Biol. 2022 ;2546 27-34
      Acylcarnitines are formed in the mitochondria by esterification between carnitine and acyl-CoAs. This occurs enzymatically via carnitine acyltransferases. Specific acylcarnitines accumulate as a result of various organic acidurias and fatty acid oxidation disorders, and, thus, acylcarnitines profiles are used for the diagnosis of these disorders. Acylcarnitines monitoring can also be used for the follow-up of patients with these disorders. Tandem mass spectrometry (MS/MS) is the most commonly used method for the analysis of acylcarnitines. An MS/MS method for the quantification of a number of acylcarnitines is described. The method involves butylation of acylcarnitines using acidified butanol. Butylated acylcarnitines are analyzed using flow injection and precursor ion scan. Multiple-reaction monitoring (MRM) is used for the analysis of low-molecular-weight acylcarnitines.
    Keywords:  Fatty acid oxidation defects; Inborn error of metabolism; Inherited metabolic disorders; Medium-chain acyl-CoA dehydrogenase deficiency; Organic acidemia; Organic acidurias; Tandem mass spectrometry
  8. Methods Mol Biol. 2023 ;2571 105-114
      The simultaneous analysis of cationic and anionic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) has been considered challenging, as often two different analytical methods are required. Although CE-MS methods for cationic metabolite profiling have already shown good performance metrics, the profiling of anionic metabolites often results in relatively low sensitivity and poor repeatability caused by problems related to unstable electrospray and corona discharge when using reversed CE polarity and detection by MS in negative ionization mode. In this protocol, we describe a chemical derivatization procedure that provides a permanent positive charge to acidic metabolites, thereby allowing us to profile anionic metabolites by CE-MS using exactly the same separation conditions as employed for the analysis of basic metabolites. The utility of the overall approach is demonstrated for the analysis of energy metabolism-related metabolites in low numbers of HepG2 cells.
    Keywords:  Capillary electrophoresis; Carboxylic acid metabolites; Chemical derivatization; HepG2 cells; Metabolomics
  9. Bone Rep. 2022 Dec;17 101620
      Amino acid metabolism regulates essential cellular functions, not only by fueling protein synthesis, but also by supporting the biogenesis of nucleotides, redox factors and lipids. Amino acids are also involved in tricarboxylic acid cycle anaplerosis, epigenetic modifications, next to synthesis of neurotransmitters and hormones. As such, amino acids contribute to a broad range of cellular processes such as proliferation, matrix synthesis and intercellular communication, which are all critical for skeletal cell functioning. Here we summarize recent work elucidating how amino acid metabolism supports and regulates skeletal cell function during bone growth and homeostasis, as well as during skeletal disease. The most extensively studied amino acid is glutamine, and osteoblasts and chondrocytes rely heavily on this non-essential amino acid during for their functioning and differentiation. Regulated by lineage-specific transcription factors such as SOX9 and osteoanabolic agents such as parathyroid hormone or WNT, glutamine metabolism has a wide range of metabolic roles, as it fuels anabolic processes by producing nucleotides and non-essential amino acids, maintains redox balance by generating the antioxidant glutathione and regulates cell-specific gene expression via epigenetic mechanisms. We also describe how other amino acids affect skeletal cell functions, although further work is needed to fully understand their effect. The increasing number of studies using stable isotope labelling in several skeletal cell types at various stages of differentiation, together with conditional inactivation of amino acid transporters or enzymes in mouse models, will allow us to obtain a more complete picture of amino acid metabolism in skeletal cells.
    Keywords:  Amino acids; Cell metabolism; Chondrocyte; Glutamine; Osteoblast; Osteoclast
  10. Methods Mol Biol. 2022 ;2546 35-43
      We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a clinical diagnostic test. Determination of SAM/SAH in plasma (20 μL) was performed by high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards (2H3-SAM and 2H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 μL sample with 180 μL of internal standard solution consisting of heavy-isotope-labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3 μL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP® (Sciex). Chromatographic separation was achieved on a 250 mm × 2.0 mm EZ-faast column from Phenomenex. Samples were eluted at a flow rate of 0.20 mL/min with a binary gradient with a total run time of 10 min. The source operated in positive ion mode at an ion spray voltage of +5000 V. SAM and SAH resolved by a gradient to 100% methanol with retention times of 5.8 and 5.5 min, respectively. HPLC chromatographic conditions did not produce complete separation of SAM and SAH, but they were completely discerned by their different fragmentation pattern in the mass spectrometer working in the MS-MS mode. The observed m/z values of the fragment ions were m/z 399→250 for SAM, m/z 385→136 for SAH, m/z 402→250 for 2H3-SAM, and m/z 203→46. The calibration curve was linear over the range of 12.5-5000 nmol/L for SAM and SAH.
    Keywords:  Mass spectrometry; Methylation; S-adenosylhomocysteine; S-adenosylmethionine
  11. Br J Cancer. 2022 Sep 17.
      BACKGROUND: Cellular metabolism is an integral component of cellular adaptation to stress, playing a pivotal role in the resistance of cancer cells to various treatment modalities, including radiotherapy. In response to radiotherapy, cancer cells engage antioxidant and DNA repair mechanisms which mitigate and remove DNA damage, facilitating cancer cell survival. Given the reliance of these resistance mechanisms on amino acid metabolism, we hypothesised that controlling the exogenous availability of the non-essential amino acids serine and glycine would radiosensitise cancer cells.METHODS: We exposed colorectal, breast and pancreatic cancer cell lines/organoids to radiation in vitro and in vivo in the presence and absence of exogenous serine and glycine. We performed phenotypic assays for DNA damage, cell cycle, ROS levels and cell death, combined with a high-resolution untargeted LCMS metabolomics and RNA-Seq.
    RESULTS: Serine and glycine restriction sensitised a range of cancer cell lines, patient-derived organoids and syngeneic mouse tumour models to radiotherapy. Comprehensive metabolomic and transcriptomic analysis of central carbon metabolism revealed that amino acid restriction impacted not only antioxidant response and nucleotide synthesis but had a marked inhibitory effect on the TCA cycle.
    CONCLUSION: Dietary restriction of serine and glycine is a viable radio-sensitisation strategy in cancer.
  12. Int J Mol Sci. 2022 Sep 18. pii: 10908. [Epub ahead of print]23(18):
      Metabolomics is a potential approach to paving new avenues for clinical diagnosis, molecular medicine, and therapeutic drug monitoring and development. The conventional metabolomics analysis pipeline depends on the data-independent acquisition (DIA) technique. Although powerful, it still suffers from stochastic, non-reproducible ion selection across samples. Despite the presence of different metabolomics workbenches, metabolite identification remains a tedious and time-consuming task. Consequently, sequential windowed acquisition of all theoretical MS (SWATH) acquisition has attracted much attention to overcome this limitation. This article aims to develop a novel SWATH platform for data analysis with a generation of an accurate mass spectral library for metabolite identification using SWATH acquisition. The workflow was validated using inclusion/exclusion compound lists. The false-positive identification was 3.4% from the non-endogenous drugs with 96.6% specificity. The workflow has proven to overcome background noise despite the complexity of the SWATH sample. From the Human Metabolome Database (HMDB), 1282 compounds were tested in various biological samples to demonstrate the feasibility of the workflow. The current study identified 377 compounds in positive and 303 in negative modes with 392 unique non-redundant metabolites. Finally, a free software tool, SASA, was developed to analyze SWATH-acquired samples using the proposed pipeline.
    Keywords:  MS/MS library; SWATH; data-independent acquisition; high-resolution LC-MS; metabolomics
  13. Methods Mol Biol. 2023 ;2576 9-19
      Extraction and quantification of endocannabinoids from biological tissues is essential to unravel their changes under physiological and pathophysiological conditions. We describe here an analytical protocol for the extraction of endocannabinoids, anandamide (archidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and endocannabinoid-like lipids such as palmitoyl ethanolamide (PEA) and oleoyl ethanolamide (OEA), as well as arachidonic acid (AA) from biological tissues using liquid-liquid extraction method and simultaneous quantification by liquid chromatography multiple reaction monitoring (LC/MRM).
    Keywords:  Endocannabinoid-like lipids; Endocannabinoids; Lipid extraction; Lipidomics; Liquid-chromatography; Multiple reaction monitoring
  14. J Lipid Res. 2022 Sep 14. pii: S0022-2275(22)00114-6. [Epub ahead of print] 100281
      Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I (HSAN1), resulting in the synthesis and accumulation of 1-deoxysphingolipids. These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxysphingolipid synthesis, they impact numerous other metabolic pathways important for cancer cells. Here we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxysphingolipid toxicity in cancer cells. We determined that both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxysphingolipid synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxysphingolipid synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.
    Keywords:  1-deoxy(dihydro)ceramide; 1-deoxysphingolipid accumulation; Metabolism; RAB5; SPT promiscuity; alanine; mitochondrial stress; serine; serine palmitoyltransferase; soft agar
  15. Metabolites. 2022 Aug 28. pii: 807. [Epub ahead of print]12(9):
      Pre-column fluorescent derivatization has been used for the fast quantification of amino acids using high-performance liquid chromatography (HPLC) systems. However, it generally requires an offline in-vial derivatization process with multiple derivatization reagents. The offline derivatization requires the same number of reaction vials as the number of sample vials for use as a reaction chamber for the derivatization reaction in an autosampler. Therefore, the number of samples analyzed per batch using the pre-column derivatization method is halved. To benefit from the pre-column derivatization method, we transformed the derivatization process from an offline chamber process to an online in-needle process (in-needle Pre-column Derivatization for Amino acids Quantification; iPDAQ). Fluorescent derivatization in the injection needle obviated the need for vacant vials as reaction chambers. Consequently, the throughput per batch improved up to two times, and the consumption of derivatization reagents was reduced to less than one-tenth of that in the conventional vial method. We demonstrated to separate and quantify the amino acids in various biological samples. Herein, we presented a novel HPLC-based amino acid quantification method that enables the continuous analysis of a large number of samples. The iPDAQ facilitates accurate amino acid quantification due to the automation of derivatization and achieves improvement in the throughput and reduction of analysis labor.
    Keywords:  HPLC; SRM1950; amino acids quantification; pre-column derivatization
  16. Methods Mol Biol. 2022 ;2522 255-266
      The development of mass spectrometry (MS)-based proteomics methods has been critical in providing new insight about cellular processes and adaptations in all domains of life. While traditional MS-based methods are not inherently quantitative, technologies are now available to overcome this limitation. Of note, stable isotope labeling of amino acids in cell culture (SILAC) is reported as a reliable tool to label proteomes for quantitative MS-based proteomics that is accurate and flexible for multiplexing. The isotopically labeled lysine and arginine are easily incorporated into the proteome of cells auxotrophic for these amino acids. Microorganisms of the domain Archaea provide a fascinating alternative to understanding cellular adaptations and responses to environmental stresses. However, the availability of preferred SILAC-based quantitative analyses is limited. This protocol describes the use of SILAC to quantitatively analyze the proteome of Haloferax volcanii, a mesophilic halophilic archaeon that is easy to grow and requires no special equipment to maintain.
    Keywords:  Archaea; Haloferax volcanii; Mass spectrometry; Oxidative stress; Proteomics; SILAC
  17. Methods Mol Biol. 2022 ;2546 295-309
      Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Measurement of MMA in biological samples is complicated because of the presence of succinic acid (SA), isomer of MMA. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for MMA. The method utilizes derivatization and positive ion mode ionization, which is specific to polycarboxylic acids (MMA and SA are dicarboxylic acids), while derivatives of monocarboxylic acids at these conditions are not ionizable and not detectable. The only organic acid, other than MMA, that is detected in this method is SA. The described method does not require chromatographic resolution of the peaks of MMA and SA; quantitative measurement of MMA is performed using a deconvolution algorithm, which mathematically resolves signal corresponding to MMA, from the combined signal of MMA/SA. Because of the high selectivity of detection, this method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features allow high-throughput analysis of MMA with injection-to-injection cycle time of approximately 1 minute.
    Keywords:  Data analysis; Deconvolution; Derivatization; Isomers; Liquid chromatography; Methylmalonic acid; Succinic acid; Tandem mass spectrometry
  18. Methods Mol Biol. 2023 ;2571 207-239
      Metabolomics is the latest of the omics sciences. It attempts to measure and characterize metabolites-small chemical compounds <1500 Da-on cells, tissue, or biofluids, which are usually products of biological reactions. As metabolic reactions are closer to the phenotype, metabolomics has emerged as an attractive science for various areas of research, including personalized medicine. However, due to the complexity of data obtained and the absence of curated databases for metabolite identification, data processing is the major bottleneck in this area since most technicians lack the required bioinformatics expertise to process datasets in a reliable and fast manner. The aim of this chapter is to describe the available tools for data processing that makes an inexperienced researcher capable of obtaining reliable results without having to undergo through huge parametrization steps.
    Keywords:  Data processing; Liquid chromatography; Mass spectrometry; Untargeted metabolomics
  19. Methods Mol Biol. 2022 ;2546 253-260
      We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) lactate as a clinical diagnostic test. Lactate is produced from cellular metabolism, primarily in muscle cells, and provides a source of energy especially during instances of low oxygen levels. Measurement of lactate in CSF provides diagnostic information regarding the body's oxidative metabolism including diagnosis of lactate acidosis, aiding in the diagnosis of blood-brain barrier glucose transporter defect and differentiation between bacterial and viral meningitis. Determination of lactate in CSF (20 μL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Lactate in CSF is determined by a 1:10 dilution with internal standard (sodium lacate-d3) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a six-point standard curve (0.625-20 mM) and has an analytical measurement range of 0.3-20 mM.
    Keywords:  Glucose transporter defect; Lactate; Lactate acidosis; Mass spectrometry
  20. Methods Mol Biol. 2023 ;2576 49-66
      The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.
    Keywords:  Docosahexaenoylethanolamide; Eicosapentaenoylethanolamide; Endocannabinoids; LC–MS; Solid phase extraction; n-3 fatty acid
  21. Methods Mol Biol. 2022 ;2546 55-64
      Analysis of clinically relevant amino acids using ion exchange chromatography coupled to photometric/fluorescent detection has been an indispensable component in the detection of inborn errors of metabolism for six decades. Detection of amino acids using mass spectrometry offers advantages in speed and analytic specificity. Employing methanol extraction and controlled butylation, C8 reversed-phase chromatography, and MS/MS detection, 32 amino acids are quantified in 20 min with clinically appropriate imprecision in plasma, urine, and cerebrospinal fluid (CSF). Quantitation is linear to 2500 μM, and limits of detection are at least 1.0 μM. Important isobaric amino acids are distinguished by chromatography or by unique patterns of fragmentation following collision-induced dissociation (CID). The technique employs commercially available reagents and may be expanded and customized for specific clinical or research settings.
    Keywords:  Amino acid; Butylation; Isobar; Liquid chromatography; Mass spectrometry; Multiple reaction monitoring; Newborn screening
  22. Methods Mol Biol. 2022 ;2546 311-319
      We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folate and is involved in the regulation of homocysteine and numerous methylation reactions, including synthesis of neurotransmitters, lipids, DNA, and RNA. Measurement of 5-MTHF in CSF provides diagnostic information regarding disorders affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism and cerebral folate deficiency. Determination of 5-MTHF in CSF (50 μL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-13C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.
    Keywords:  5-Methyltetrahydrofolate; Cerebral folate deficiency; Mass spectrometry; Methylation
  23. STAR Protoc. 2022 Sep 22. pii: S2666-1667(22)00595-0. [Epub ahead of print]3(4): 101715
      Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites.
    Keywords:  Chemistry; Immunology; Mass spectrometry; Metabolism; Metabolomics
  24. Methods Mol Biol. 2023 ;2571 57-69
      Metabolomics, alone or in combination with other omics sciences, has shown great relevance in a large number of investigations in different branches of biomedicine, often providing novel discoveries and helping to expand the knowledge. Metabolomics analyses are carried out using different techniques, but in this chapter, we focus on liquid chromatography coupled to high-resolution mass spectrometry. The designated methodology consists of an untargeted approach for the analysis of plasma samples. The use of this method, with a reverse-phase column and electrospray ionization in positive mode, covers the detection of a broad range of metabolites, mainly of nonpolar and of intermediate polarity. This chapter also reviews the mass fragmentation spectra for the identification of bile acids, acylcarnitines, and glycerophospholipids.
    Keywords:  Acylcarnitines; Bile acids; Glycerophospolipids; Lipids; Mass spectrometry; Plasma; Reverse phase; Untargeted metabolomics
  25. Front Oncol. 2022 ;12 991051
      Pancreatic cancer (PC) is burdened with a low 5-year survival rate and high mortality due to a severe lack of early diagnosis methods and slow progress in treatment options. To improve clinical diagnosis and enhance the treatment effects, we applied metabolomics using ultra-high-performance liquid chromatography with a high-resolution mass spectrometer (UHPLC-HRMS) to identify and validate metabolite biomarkers from paired tissue samples of PC patients. Results showed that the metabolic reprogramming of PC mainly featured enhanced amino acid metabolism and inhibited sphingolipid metabolism, which satisfied the energy and biomass requirements for tumorigenesis and progression. The altered metabolism results were confirmed by the significantly changed gene expressions in PC tissues from an online database. A metabolites biomarker panel (six metabolites) was identified for the differential diagnosis between PC tumors and normal pancreatic tissues. The panel biomarker distinguished tumors from normal pancreatic tissues in the discovery group with an area under the curve (AUC) of 1.0 (95%CI, 1.000-1.000). The biomarker panel cutoff was 0.776. In the validation group, an AUC of 0.9000 (95%CI = 0.782-1.000) using the same cutoff, successfully validated the biomarker signature. Moreover, this metabolites panel biomarker had a great capability to predict the overall survival (OS) of PC. Taken together, this metabolomics method identifies and validates metabolite biomarkers that can diagnose the onsite progression and prognosis of PC precisely and sensitively in a clinical setting. It may also help clinicians choose proper therapeutic interventions for different PC patients and improve the survival of PC patients.
    Keywords:  Genotype-Tissue Expression (GTEx); The Cancer Genome Atlas (TCGA); biomarker; metabolism; pancreatic cancer; prognosis
  26. Antioxidants (Basel). 2022 Aug 29. pii: 1692. [Epub ahead of print]11(9):
      Glucose and glutamine play a crucial role in the metabolic reprogramming of cancer cells. Proliferating cells metabolize glucose in the aerobic glycolysis for energy supply, and glucose and glutamine represent the primary sources of carbon atoms for the biosynthesis of nucleotides, amino acids, and lipids. Glutamine is also an important nitrogen donor for the production of nucleotides, amino acids, and nicotinamide. Several membrane receptors strictly control metabolic reprogramming in cancer cells and are considered new potential therapeutic targets. Formyl-peptide receptor 2 (FPR2) belongs to a small family of GPCRs and is implicated in many physiopathological processes. Its stimulation induces, among other things, NADPH oxidase-dependent ROS generation that, in turn, contributes to intracellular signaling. Previously, by phosphoproteomic analysis, we observed that numerous proteins involved in energetic metabolism are uniquely phosphorylated upon FPR2 stimulation. Herein, we investigated the role of FPR2 in cell metabolism, and we observed that the concentrations of several metabolites associated with the pentose phosphate pathway (PPP), tricarboxylic acid cycle, nucleotide synthesis, and glutamine metabolism, were significantly enhanced in FPR2-stimulated cells. In particular, we found that the binding of specific FPR2 agonists: (i) promotes NADPH production; (ii) activates the non-oxidative phase of PPP; (iii) induces the expression of the ASCT2 glutamine transporter; (iv) regulates oxidative phosphorylation; and (v) induces the de novo synthesis of pyrimidine nucleotides, which requires FPR2-dependent ROS generation.
    Keywords:  NADPH oxidase; formyl-peptide receptors; glucose metabolism; glutamine transporter; metabolic reprogramming; synthesis of pyrimidine nucleotides
  27. Methods Mol Biol. 2023 ;2571 133-142
      Metabolomics continues to progress, but obstacles remain. The preservation of metabolites in the target tissue and gathering information on the current metabolic state of the organism of interest proves challenging. Robustness, reproducibility, and reliable quantification are necessary for confident metabolite identification and should always be considered for effective biomarker discovery. Recent advancements in analytical platforms, techniques, and data analysis make metabolomics a promising omics for significant research. However, there is no single approach to effectively capturing the metabolome. Coupling separation techniques may improve the power of the analysis and facilitate confident metabolite identification, especially when performing untargeted metabolomics. In this chapter, we will present an untargeted metabolomic analysis of brain tissue from C57BL/6 mice using two UHPLC-MS methods based on reversed-phase and HILIC chromatography.
    Keywords:  HILIC chromatography; High-resolution mass spectrometry; LC–MS/MS; Reversed-phase chromatography; Ultrahigh-performance liquid chromatography; Untargeted metabolomics
  28. Methods Mol Biol. 2022 ;2546 271-284
      Plasma lysosphingolipids are highly elevated in patients with Gaucher, Krabbe, Fabry, and Niemann-Pick diseases and tend to accumulate to a greater extent than their respective primary sphingolipids in the plasma of affected patients. In this chapter, we describe two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to measure plasma concentrations of four lysosphingolipids species. The first method described measures glucosylsphingosine (lyso-GL1) and galactosylsphingosine (psychosine), biomarkers that accumulate in Gaucher and Krabbe diseases, respectively. The second method measures globotriaosylsphingosine (lyso-Gb3) and sphingosylphosphorylcholine (lyso-SPM), biomarkers for Fabry and Niemann-Pick diseases, respectively. Each method utilizes isotope-labeled internal standards and multipoint calibration curves to quantify the analytes of interest. Briefly, plasma samples are mixed with five volumes of LC-MS grade methanol containing internal standard, and protein is removed via centrifugation. Supernatant is dried and resuspended in initial mobile phase. Samples are separated by liquid chromatography using either a BEH amide column (lyso-GL1 + psychosine) or a C18 column (lyso-Gb3 + lyso-SPM). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. Using these methods, we have observed elevations of these lyso- species in Gaucher, Fabry, and Niemann-Pick and successfully distinguished different subtypes reflecting the disease severity.
    Keywords:  Fabry disease; Galactosylsphingosine (psychosine); Gaucher disease; Globotriaosylsphingosine (lyso-Gb3); Glucosylsphingosine (lyso-GL1); Krabbe disease; Liquid chromatography; Mass spectrometry; Niemann–Pick disease; Sphingosylphosphorylcholine (lyso-SPM)
  29. Front Immunol. 2022 ;13 937406
      The tumor microenvironment (TME) has become a major research focus in recent years. The TME differs from the normal extracellular environment in parameters such as nutrient supply, pH value, oxygen content, and metabolite abundance. Such changes may promote the initiation, growth, invasion, and metastasis of tumor cells, in addition to causing the malfunction of tumor-infiltrating immunocytes. As the neoplasm develops and nutrients become scarce, tumor cells transform their metabolic patterns by reprogramming glucose, lipid, and amino acid metabolism in response to various environmental stressors. Research on carcinoma metabolism reprogramming suggests that like tumor cells, immunocytes also switch their metabolic pathways, named "immunometabolism", a phenomenon that has drawn increasing attention in the academic community. In this review, we focus on the recent progress in the study of lipid metabolism reprogramming in immunocytes within the TME and highlight the potential target molecules, pathways, and genes implicated. In addition, we discuss hypoxia, one of the vital altered components of the TME that partially contribute to the initiation of abnormal lipid metabolism in immune cells. Finally, we present the current immunotherapies that orchestrate a potent antitumor immune response by mediating the lipid metabolism of immunocytes, highlight the lipid metabolism reprogramming capacity of various immunocytes in the TME, and propose promising new strategies for use in cancer therapy.
    Keywords:  immunocyte; immunometabolism; immunotherapy; lipid metabolism reprogramming; tumor microenvironment
  30. Methods Mol Biol. 2022 ;2546 195-204
      Serotonin (5-hydroxytryptamine) is a neurotransmitter produced in excess by carcinoid tumors, which develop from enterochromaffin cells. 5-Hydroxyindoleacetic acid (5-HIAA) is the primary urinary metabolite of serotonin, making measurement of 5-HIAA useful in the diagnosis and management of carcinoid tumors. Here we describe a simple, inexpensive, and fast method for the detection and quantification of 5-HIAA in urine. Samples are prepared by simple 1:1 dilution. The instrumental analysis is performed by chromatographic separation on a reverse-phase analytical column followed by detection using a triple quadrupole mass spectrometer with electrospray ionization in positive ion mode. Data are acquired by multiple reaction monitoring (MRM).
    Keywords:  5-Hydroxyindolacetic acid; Carcinoid tumor; Tandem mass spectrometry; Ultra-performance liquid chromatography
  31. Methods Mol Biol. 2022 ;2546 509-521
      Peroxisomal disorders are a heterogeneous group of genetic disorders caused by impaired peroxisomal biogenesis or by defects in single peroxisomal proteins. The most common peroxisomal disorders are Zellweger spectrum disorders (ZSDs), due to pathogenic variants in one of the 13 PEX genes, and X-linked adrenoleukodystrophy/adrenomyeloneuropathy (X-ALD/AMN), due to pathogenic variants in ATP-binding cassette transporter type D1 (ABCD1) gene. Peroxisomes perform multiple essential cellular functions, including β-oxidation of very-long-chain fatty acids (VLCFAs), pristanic acid and some bile acid intermediates, and α-oxidation of phytanic acid. In most patients, abnormal levels of VLCFAs and/or branched-chain fatty acids (BCFAs, e.g., phytanic and pristanic acids) are present; hence, measuring these analytes is critical when suspecting a peroxisomal disorder. This chapter describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify VLCFAs and BCFAs in plasma or serum for the diagnosis of peroxisomal disorders. The method consists of an acid hydrolysis step to release the fatty acids from their coenzyme A esters followed by derivatization using oxalyl chloride, dimethylaminoethanol, and then methyl iodide. The trimethyl-amino-ethyl (TMAE) iodide ester derivatives are analyzed using UPLC-MS/MS in positive electrospray ionization and multiple reaction-monitoring (MRM) mode. Quantitation is performed using a five-point calibration curve after normalizing with deuterated internal standards.
    Keywords:  Branched-chain fatty acids; Liquid chromatography-tandem mass spectrometry; Peroxisomal disorders; Very-long-chain fatty acids
  32. Methods Mol Biol. 2022 ;2546 175-183
      Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are catecholamine metabolites used in the diagnostic workup of neuroendocrine tumors. Here we describe a simple dilute-and-shoot method for simultaneously quantitating HVA and VMA in human urine specimens. The method employs analyte separation on a reverse-phase liquid chromatography column followed by detection using electrospray ionization triple quadrupole mass spectrometry (ESI-MS/MS), wherein qualifier and quantifier ion transitions are monitored. This is a simple and fast analytical method with an injection-to-injection time of 4 min.
    Keywords:  Adrenal medulla; Homovanillic acid; Neuroblastoma; Pheochromocytoma; Vanillylmandelic acid
  33. Methods Mol Biol. 2022 ;2522 287-300
      Despite the ecological, evolutionary and economical significance of archaea, key aspects of their cell biology, metabolic pathways, and adaptations to a wide spectrum of environmental conditions, remain to be elucidated. Proteomics allows for the system-wide analysis of proteins, their changes in abundance between different conditions, as well as their post-translational modifications, providing detailed insights into the function of proteins and archaeal cell biology. In this chapter, we describe a sample preparation and mass spectrometric analysis workflow that has been designed for Haloferax volcanii but can be applied to a broad range of archaeal species. Furthermore, proteomics experiments provide a wealth of data that is invaluable to various disciplines. Therefore, we previously initiated the Archaeal Proteome Project (ArcPP), a community project that combines the analysis of multiple datasets with expert knowledge in various fields of archaeal research. The corresponding bioinformatic analysis, allowing for the integration of new proteomics data into the ArcPP, as well as the interactive exploration of ArcPP results is also presented here. In combination, these protocols facilitate an optimized, detailed and collaborative approach to archaeal proteomics.
    Keywords:  Archaea; Bioinformatics; Fractionation; Mass spectrometry; Post-translational modifications; Protein database search; Proteomics
  34. Methods Mol Biol. 2022 ;2546 165-174
      We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of CSF GABA has clinical utility in diagnosing inborn errors of GABA metabolism, specifically for deficiencies of GABA-transaminase and succinic semialdehyde dehydrogenase. Quantitation of CSF GABA is performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Quantitation of total GABA in CSF requires additional sample preparation in order to hydrolyze all the conjugated GABA in the sample to free GABA. Complete hydrolysis is performed incubating sample at >100 °C in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90% acetonitrile/0.1% formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 to 1000 nM and 0.63 to 80 μM for free and total GABA, respectively.
    Keywords:  Cerebrospinal fluid; GABA; Mass spectrometry; Seizures
  35. Methods Mol Biol. 2022 ;2546 95-104
      L-carnitine is a crucial component for transporting long-chained fatty acids from the cytosol into the mitochondrial matrix for fatty acid oxidation. During this process, carnitine forms numerous acylcarnitines before being recycled into the cytosol. Abnormal levels of free carnitine, total carnitine, and acylcarnitines in serum can be indicative of a metabolic disorder before symptoms are present. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is described for the determination of free and total carnitine in serum. To measure total carnitine, samples are spiked with deuterated carnitine (internal standard) and hydrolyzed with potassium hydroxide to convert acylcarnitines to carnitine. The reaction is quenched by the addition of hydrochloric acid. Carnitine is extracted via a methanolic protein precipitation. The solution is then injected on LC-MS/MS for analysis to determine the carnitine concentration using multiple-reaction monitoring.
    Keywords:  Acylcarnitine; Carnitine; Fatty acid oxidation; Tandem mass spectrometry
  36. Methods Mol Biol. 2022 ;2546 83-94
      Acylcarnitines are formed when an acyl group is transferred from coenzyme A to a molecule of L-carnitine. In organic acidemias, and in fatty acid oxidation disorders, specific acylcarnitine species accumulate in a pattern that is characteristic for each disease. For this reason, acylcarnitine analysis is widely used for screening and diagnosis of inherited disorders of metabolism. The most common method for acylcarnitine analysis uses flow injection tandem mass spectrometry. Flow injection analysis allows for high throughput, however, does not provide separation of isomeric and isobaric compounds. Among the acylcarnitine species which can be affected by the presence of isomeric/isobaric compounds, C4-carnitine and C5DC-carnitine are probably the ones encountered most often. The method presented here is performed on urine and utilizes butanolic HCL to derivatize acylcarnitines, ultra-performance liquid chromatography to resolve C4- and C5-DC isomers and isobars, and quantitation of these species using multiple-reaction monitoring (MRM).
    Keywords:  Butyrylcarnitine; Fatty acid oxidation disorders; Glutaric acidemia type I; Glutarylcarnitine; Isobutyryl-CoA dehydrogenase deficiency; Isobutyrylcarnitine; Organic acidemia; Short-chain acyl-CoA dehydrogenase (SCAD) deficiency
  37. Methods Mol Biol. 2022 ;2546 217-226
      Administration of exogenous insulin or insulin analogs is a common cause of hypoglycemia. The etiology of hypoglycemic episodes can be investigated by the measurement of insulin. However, frequently used synthetic insulin analogs show variable reactivity with immunoassays designed for the quantification of human insulin and may produce misleading results. To overcome this challenge, mass spectrometric methods can be applied to differentiate and accurately quantify insulin and its analogs. Here we describe a liquid chromatography-tandem high-resolution accurate mass (LC-HRAM) for the highly specific and independent quantification of insulin and its synthetic analogs including aspart, detemir, glargine, glulisine, and lispro. This method utilizes antibody affinity extraction followed by analysis on a high-resolution accurate mass spectrometer.
    Keywords:  Hyperinsulinemia; Hypoglycemia; Insulin analogs; Liquid chromatography–high-resolution mass spectrometry
  38. Cancers (Basel). 2022 Sep 19. pii: 4526. [Epub ahead of print]14(18):
      Obesity is a prominent risk factor for certain types of tumor progression. Adipocytes within tumor stroma contribute to reshaping tumor microenvironment (TME) and the metabolism and metastasis of tumors through the production of cytokines and adipokines. However, the crosstalk between adipocytes and tumor cells remains a major gap in this field. Known as a subtype of selective autophagy, lipophagy is thought to contribute to lipid metabolism by breaking down intracellular lipid droplets (LDs) and generating free fatty acids (FAs). The metastatic potential of cancer cells closely correlates with the lipid degradation mechanisms, which are required for energy generation, signal transduction, and biosynthesis of membranes. Here, we discuss the recent advance in the understanding of lipophagy with tumor lipid metabolism and review current studies on the roles of lipoghagy in the metastasis of certain human malignancies. Additionally, the novel candidate drugs targeting lipophagy are integrated for effective treatment strategies.
    Keywords:  cancer metastasis; lipid metabolism; lipophagy
  39. Methods Mol Biol. 2022 ;2546 119-128
      Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing's syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement.We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotope-labeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection.
    Keywords:  APCI; Cortisone; Cushing’s syndrome; LC-MS/MS; Urinary free cortisol
  40. Cell Death Dis. 2022 Sep 21. 13(9): 808
      Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer that displays highly aggressive with poor prognosis. Owing to the limited targets and drugs for TNBC clinical therapy, it is necessary to investigate the factors regulating cancer progression and develop novel therapies for cancer treatment. Ferroptosis, a nonapoptotic form of programmed cell death characterized by accumulation of iron-dependent peroxidation of phospholipids, is regulated by cellular metabolism, redox homeostasis, and various cancer-related signaling pathways. Recently, considerable progress has been made in demonstrating the critical role of lipid metabolism in regulating ferroptosis, indicating potential combinational therapeutic strategies for cancer treatment. In this study, by drug combination screen of lipid metabolism compounds with ferroptosis inducers in decreasing TNBC cell viability, we found potent synergy of the CB1 antagonist rimonabant with erastin/(1 S, 3 R)-RSL3 (RSL3) in inhibiting TNBC cell growth both in vitro and in vivo via promoting the levels of lipid peroxides, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and cytosolic reactive oxygen species (ROS) production, enhancing intracellular glutathione (GSH) depletion and inducing G1 cell cycle arrest. We identified that inhibition of CB1 promoted the effect of erastin/RSL3 on inducing ferroptosis and enhanced their inhibitory effect on tumor growth. Using RNA-Seq, fatty acid analyses and functional assays, we found that CB1 regulated stearoyl-CoA desaturase 1 (SCD1)- and fatty acyl desaturase 2 (FADS2)-dependent fatty acid metabolism via phosphatidylinositol 3 kinase (PI3K)-AKT and mitogen-activated protein kinase (MAPK) signaling pathways to modulate ferroptosis sensitivity in TNBC cells. These data demonstrate that dual targeting of CB1 and ferroptosis could be a promising therapeutic strategy for TNBC.
  41. Methods Mol Biol. 2022 ;2546 523-537
      Liquid-chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be an effective approach in the clinical analysis of 25-OH-vitamin D in patient serum. Test volumes vary among laboratories and different levels of throughput are required for different settings. LC-MS/MS assays with multiple LC channels can be beneficial for labs with the demand of large sample volume (e.g., 300 or more samples) to control costs and fulfill a reasonable turnaround time. We hereby present an assay that employs 4 LC channels (4-plex), which are coupled to the TSQ Endura triple-quadrupole (QqQ) MS instrument, for a high-throughput solution. Briefly, the pre- and postelution segments of the LC gradient are diverted to waste via solenoid valve controls, reserving the data acquisition for only the elution segment per injection per channel at a time. The multiplexing affords a manifold increase in throughput and the optimization of the duty cycle, without compromise in assay performance and precision.
    Keywords:  25-OH-vitamin D; LC-to-LC channel precision; Multiple-reaction-monitoring (MRM); atmospheric pressure chemical ionization (APCI); duty cycle; high-throughput LC-MS/MS; multiplex HPLC
  42. PLoS One. 2022 ;17(9): e0274623
      Three-dimensional (3D) cell culture technologies, which more closely mimic the complex microenvironment of tissue, are being increasingly evaluated as a tool for the preclinical screening of clinically promising new molecules, and studying of tissue metabolism. Studies of metabolites released into the extracellular space (secretome) allow understanding the metabolic dynamics of tissues and changes caused by therapeutic interventions. Although quite advanced in the field of proteomics, studies on the secretome of low molecular weight metabolites (< 1500 Da) are still very scarce. We present an untargeted metabolomic protocol based on the hybrid technique of liquid chromatography coupled with high-resolution mass spectrometry for the analysis of low-molecular-weight metabolites released into the culture medium by 3D cultures and co-culture (secretome model). For that we analyzed HT-29 human colon carcinoma cells and 3T3-L1 preadipocytes in 3D-monoculture and 3D-co-culture. The putative identification of the metabolites indicated a sort of metabolites, among them arachidonic acid, glyceric acid, docosapentaenoic acid and beta-Alanine which are related to cancer and obesity. This protocol represents a possibility to list metabolites released in the extracellular environment in a comprehensive and untargeted manner, opening the way for the generation of metabolic hypotheses that will certainly contribute to the understanding of tissue metabolism, tissue-tissue interactions, and metabolic responses to the most varied interventions. Moreover, it brings the potential to determine novel pathways and accurately identify biomarkers in cancer and other diseases. The metabolites indicated in our study have a close relationship with the tumor microenvironment in accordance with the literature review.
  43. Genes (Basel). 2022 Sep 03. pii: 1585. [Epub ahead of print]13(9):
      Even though breast cancer is the most diagnosed cancer among women, treatments are not always successful in preventing its progression. Recent studies suggest that hypoxia and the extracellular matrix (ECM) are important in altering cell metabolism and tumor metastasis. Therefore, the aim of this review is to study the crosstalk between hypoxia and the ECM and to assess their impact on breast cancer progression. The findings indicate that hypoxic signaling engages multiple mechanisms that directly contribute to ECM remodeling, ultimately increasing breast cancer aggressiveness. Second, hypoxia and the ECM cooperate to alter different aspects of cell metabolism. They mutually enhance aerobic glycolysis through upregulation of glucose transport, glycolytic enzymes, and by regulating intracellular pH. Both alter lipid and amino acid metabolism by stimulating lipid and amino acid uptake and synthesis, thereby providing the tumor with additional energy for growth and metastasis. Third, YAP/TAZ signaling is not merely regulated by the tumor microenvironment and cell metabolism, but it also regulates it primarily through its target c-Myc. Taken together, this review provides a better understanding of the crosstalk between hypoxia and the ECM in breast cancer. Additionally, it points to a role for the YAP/TAZ mechanotransduction pathway as an important link between hypoxia and the ECM in the tumor microenvironment, driving breast cancer progression.
    Keywords:  YAP/TAZ; breast cancer; cell metabolism; extracellular matrix; hypoxia; mechanotransduction; tumor microenvironment
  44. Anal Chem. 2022 Sep 22.
      Untargeted liquid chromatography/high-resolution mass spectrometry (LC/HRMS) assays in metabolomics and exposomics aim to characterize the small molecule chemical space in a biospecimen. To gain maximum biological insights from these data sets, LC/HRMS peaks should be annotated with chemical and functional information including molecular formula, structure, chemical class, and metabolic pathways. Among these, molecular formulas may be assigned to LC/HRMS peaks through matching theoretical and observed isotopic profiles (MS1) of the underlying ionized compound. For this, we have developed the Integrated Data Science Laboratory for Metabolomics and Exposomics-United Formula Annotation (IDSL.UFA) R package. In the untargeted metabolomics validation tests, IDSL.UFA assigned 54.31-85.51% molecular formula for true positive annotations as the top hit and 90.58-100% within the top five hits. Molecular formula annotations were also supported by tandem mass spectrometry data. We have implemented new strategies to (1) generate formula sources and their theoretical isotopic profiles, (2) optimize the formula hits ranking for the individual and aligned peak lists, and (3) scale IDSL.UFA-based workflows for studies with larger sample sizes. Annotating the raw data for a publicly available pregnancy metabolome study using IDSL.UFA highlighted hundreds of new pregnancy-related compounds and also suggested the presence of chlorinated perfluorotriether alcohols (Cl-PFTrEAs) in human specimens. IDSL.UFA is useful for human metabolomics and exposomics studies where we need to minimize the loss of biological insights in untargeted LC/HRMS data sets. The IDSL.UFA package is available in the R CRAN repository Detailed documentation and tutorials are also provided at
  45. Methods Mol Biol. 2023 ;2576 21-40
      Different mass spectrometric techniques have been used over the past decade to quantify endocannabinoids (eCBs) and related lipids. Even with the level of molecular fingerprinting accuracy of an instrument like the most advanced triple quadrupole mass spectrometer, if one is not getting the most optimized sample to the detector in a way that this improved technology can be of use, then advancements can be stymied. Here, our focus is on review and discussion of sample preparation methodologies used to isolate the eCB anandamide and its close congeners N-acyl ethanolamines and structural congeners (i.e., lipo amino acids, lipoamines, N-acyl amides) in biological fluids. Most of our focus will be on the analysis of these lipids in plasma/serum, but we will also discuss how the same techniques can be used for the analysis of saliva and breast milk.
    Keywords:  Acyl amino acids; Endocannabinoids; HPLC; Lipidomics; Lipoamino acids; Mass spectrometry; Solid phase extraction
  46. Methods Mol Biol. 2023 ;2571 257-269
      Mass spectrometry is a powerful analytical technique used to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules. Nevertheless, the transfer of data from one instrument to another is one of the main problems, and obtaining the same or similar information from an analogous instrument but from a different manufacturer or even with the same instrument after carrying out the analyses in different times spacing is not possible. Hence, a general methodology to provide a chromatographic signal (or chromatogram) independent of the instrument is needed. In this sense, this book chapter describes the standardization procedure of chromatographic signals obtained from mass spectrometry platforms to obtain instrument-agnostic chromatographic signals for the determination of standard retention scores. This parameter may be used for the quantification of compounds when different mass spectrometry platforms coupled to ultrahigh-performance liquid chromatography are employed.
    Keywords:  Instrument-agnostizing methodology; Instrument-independent analytical signals; Mass spectrometry; Standardized chromatogram
  47. Methods Mol Biol. 2022 ;2546 65-81
      Branched-chain amino acids (BCAA), including valine, alloisoleucine, isoleucine, and leucine, play significant roles in a number of metabolic pathways in the body. Deficiency in branched-chain ketoacid dehydrogenase complex, an enzyme required for metabolism of those amino acids, will lead to elevation and accumulation of BCAA and ketoacids in bodily fluids. This results in maple syrup urine disease (MSUD), a condition estimated to affect 1 in 100,000-300,000 births. If MSUD is not diagnosed in the first few days of life, progression of this disease can lead to intellectual disability, coma, irreversible brain damage, seizures, or even death. If diagnosed early, MSUD can be managed by monitoring the blood concentrations of BCAA and adjusting the patient's dietary intake accordingly. Therefore, it is critical to have a rapid, accurate, and reliable BCAA assay for confirmation of MSUD in newborns as well as routine monitoring of MSUD patients. Here, we describe a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for BCAA measurement which requires only 20 μL of plasma. The sample preparation does not require derivatization and only involves protein precipitation with LC/MS-grade methanol, which contains leucine(13C6;15N), isoleucine(13C6;15N), and valine(13C5;15N) as the internal standards. The final sample extracts do not require dry-down and reconstitution and are readily compatible with the liquid chromatography (LC) method. BCAA are separated using the isocratic gradient method on a mixed-mode Intrada column. Multiple-reaction monitoring (MRM) mode is used for MS/MS detection to monitor the parent-to-daughter transitions m/z 132.2 to 86.4 for leucine, isoleucine, and alloisoleucine; m/z 118.2 to 72.4 for valine; m/z 139.2 to 92.4 for leucine(13C6;15N) and isoleucine(13C6;15N); and m/z 124.2 to 77.4 for valine(13C5;15N).
    Keywords:  Branched-chain amino acid; Liquid chromatography; Maple syrup urine disease; Mass spectrometry; Plasma; Quantification
  48. Methods Mol Biol. 2022 ;2544 129-144
      Hepatocytes play an important role in maintaining homeostasis in living organisms by carrying out various metabolic functions. The urea cycle, one of the metabolic pathways taking place in hepatocytes, is an important metabolic pathway that converts toxic ammonia to nontoxic urea. Performing quantitative assessments of individual metabolite levels using a mass spectrometer is useful for assessing the metabolic state of the urea cycle in hepatocytes. In addition, metabolic flux analysis using stable isotopes and a mass spectrometer is a new technique for measuring the metabolic state. It enables conducting specific, objective, and quantitative measurement of the activated state of the target metabolic pathway regardless of external disturbing factors. This section describes the technical background and methodology of performing metabolic flux analysis of the urea cycle by mass spectrometry.
    Keywords:  Hepatocytes;  Metabolic flux analysis;  Urea cycle
  49. Methods Mol Biol. 2022 ;2546 365-373
      Parathyroid hormone (PTH), an 84-amino acid peptide hormone, is a major regulator of calcium homeostasis. Quantification of PTH in serum is used clinically to investigate calcium imbalances and for monitoring osteodystrophy in patients with renal failure. In addition to intact PTH, several PTH fragments are found in circulation. Recent studies have shown that accurate quantification of PTH fragments may provide valuable clinical information in certain scenarios. In this chapter, a high-resolution mass spectrometry-based method for quantification of PTH (1-84) and its fragments is described. This method involves immunoaffinity capture of intact PTH and PTH-fragments followed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS).
    Keywords:  Chronic kidney disease; Liquid chromatography–high-resolution mass spectrometry; PTH1–84; Parathyroid hormone; Peptide fragments, Immunopurification
  50. Methods Mol Biol. 2023 ;2571 149-155
      Aqueous humor (AH) is a transparent fluid that fills the anterior segment of the eye. The composition and level of metabolites in AH are important for understanding its physiology and changes caused by the occurrence of eye disease. A simple method for the preparation and analysis of AH samples was developed using the liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technique. The analyses were performed using two types of chromatography: reversed-phase liquid chromatography-mass spectrometry (LC-RP-MS) and hydrophilic interaction liquid chromatography-mass spectrometry (LC-HILIC-MS), in the sample prepared by one protocol.
    Keywords:  Aqueous humor; HILIC analysis; LC–MS; Reversed-phase analysis; Untargeted metabolomics
  51. Methods Mol Biol. 2023 ;2576 41-47
      LC-MS/MS is a powerful analytical technique that provides unequivocal identification and reliable quantification of the analytes, using Selected Reaction Monitoring or Multi Reaction Monitoring acquisition mode.Anandamide (N-arachidonoylethanolamine, AEA) and 2-Arachidonoylglycerol (2-AG) are the most abundant endocannabinoids (eCBs), which play a major role in a wide variety of physiological and pathological processes. Analysis of those compounds by means of LC-MS/MS allows the detection of very low concentrations in biological samples. Here, we describe how to determine AEA and 2-AG levels in tiny samples of tissues and plasma through LC-MS/MS, by using very quick and easy-to-perform extraction procedures, with reduced solvent consumption.
    Keywords:  2-AG; AEA; LC–MS/MS; Plasma; Tissue; μSPE
  52. Methods Mol Biol. 2023 ;2571 95-103
      Capillary electrophoresis-mass spectrometry (CE-MS) is gaining interest for metabolomics studies because of its high separation efficiency, selectivity, and versatility. The ability to inject nanoliters from only a few microliters of sample in the injection vial makes this approach very suited for volume-limited applications. However, the low injection volumes could compromise the detection sensitivity of CE-MS, thereby potentially limiting its scope in metabolomics. To overcome this issue, online sample preconcentration methods have been developed to increase sample-loading volumes without hampering the intrinsic high separation efficiency of CE. In this protocol, online preconcentration with sample stacking based on pH junction was assessed for the direct profiling of endogenous metabolites in rat brain microdialysates. Sample stacking was realized by a pre-injection of ammonium hydroxide, followed by a large sample injection (i.e., about 17% of the total capillary volume). It is shown that this relatively simple and fast preconcentration procedure is fully compatible with the high-salt concentration in microdialysates and significantly improves the detection sensitivity of the CE-MS method.
    Keywords:  Brain microdialysate; Capillary electrophoresis; Direct analysis; Mass spectrometry; Metabolic profiling
  53. Methods Mol Biol. 2022 ;2546 431-437
      Inborn errors of pyrimidine metabolism result from deficiencies in pyrimidine de novo synthesis, degradation, and salvage pathways. Enzymatic deficiencies in pyrimidine catabolism lead to mitochondrial neurogastrointestinal encephalopathy (MNGIE), pyrimidinuria, dihydropyrimidinuria, ureidopropionic aciduria, and other disorders. While MNGIE presents with gastrointestinal dysmotility, cachexia and leukoencephalopathy, pyrimidinuria, and dihydropyrimidinuria may show symptoms of epilepsy, autism, mental retardation, and dysmorphic features. The application of HPLC-MS/MS facilitates rapid screening of pyrimidine metabolites. Here we describe a sensitive and reliable LC-MS/MS method for quantitative determination of uracil, thymine, thymidine, dihydrouracil, and dihydrothymine in urine that are diagnostic biomarkers of MNGIE, pyrimidinuria, and dihydropyrimidinuria.
    Keywords:  Dihydropyrimidinuria; LC-MS/MS; Laboratory diagnosis; Liquid chromatography-tandem mass spectrometry; MNGIE; Nucleotides; Pyrimidine; Pyrimidinuria
  54. Methods Mol Biol. 2023 ;2576 329-348
      Endocannabinoids at nanomolar physiological concentrations cross cellular membranes by facilitated diffusion, a process that can be studied by measuring transport kinetics and endocannabinoid trafficking employing radioligands and mass spectrometry. Here, we describe radiosubstrate-based assays using arachidonoyl[1-3H]ethanolamine and 2-arachidonoyl[1,2,3-3H]glycerol to measure cellular endocannabinoid uptake in a three-phase assay with human U937 cells. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS)-based lipidomics was used to interrogate the roles of serum and albumin for endocannabinoid trafficking in U937 cells.
    Keywords:  2-Arachidonoylglycerol; Anandamide; Endocannabinoid uptake; LC–ESI–MS/MS
  55. Methods Mol Biol. 2022 ;2546 501-508
      Abnormal accumulation of very-long-chain fatty acids (VLCFAs), defined as molecules with greater than 22 carbons, and branched-chain fatty acids, pristanic and phytanic acids, is characteristic of inborn errors of peroxisomal biogenesis or function. X-linked adrenoleukodystrophy, Zellweger spectrum disorders, rhizomelic chondrodysplasia punctata, and Refsum syndrome can be diagnosed biochemically by quantitation of these metabolites in plasma. Ratios of C24/C22 and C26/C22 can help improve detection of X-linked adrenoleukodystrophy. Analysis using gas-chromatography mass spectrometry (GC/MS) after acid/base hydrolysis, organic solvent extraction, and derivatization is an established method for clinical diagnostics. This chapter describes detailed steps to process plasma samples for GC/MS analysis.
    Keywords:  GC/MS; Gas-chromatography mass spectrometry; Peroxisomal disorders; Phytanic acid; Pristanic acid; VLCFAs; Very-long-chain fatty acids
  56. Methods Mol Biol. 2022 ;2546 141-148
      Adult and pediatric endocrinology and oncology often requires measuring serum estrogens and testosterone at very low concentrations. Conventional immunoassay methods often lack the required performance to meet this analytical need, and mass spectrometry techniques must be employed. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum, utilizing commercially available calibrators and without the need for chemical derivatization. Serum samples, after the addition of an internal standard, are combined with a hexane:ethyl acetate extraction solution. The samples are vortexed, and the organic layer is decanted into a clean sample tube and evaporated to dryness under a stream of nitrogen. The samples are reconstituted in a water:methanol solution and separated chromatographically using a reversed-phase HPLC column. Subsequent mass spectrometry is performed using both positive ion mode for Te and negative ion mode for E2.
    Keywords:  Commercial calibrators; Estradiol; Liquid chromatography; Mass spectrometry; Serum; Testosterone
  57. STAR Protoc. 2022 Sep 17. pii: S2666-1667(22)00573-1. [Epub ahead of print]3(4): 101693
      Immunoprecipitation (IP) of endogenously expressed proteins is one of the most biologically relevant techniques to identify protein-protein interactions. We describe an adaptable IP protocol reliant on a specific antibody to the target protein. We detail a quantitative proteomics workflow for the unbiased identification of co-immunoprecipitating proteins, known collectively as an interactome. This includes protocols for the tryptic digestion, Tandem Mass Tag labeling and fractionation of peptides, and their identification and quantification using liquid chromatography-mass spectrometry including computational and statistical analysis. For complete details on the use and execution of this protocol, please refer to Johnson et al. (2020).
    Keywords:  Cell biology; Mass spectrometry; Protein expression and purification; Proteomics
  58. Metabolites. 2022 Aug 24. pii: 779. [Epub ahead of print]12(9):
      Liver diseases cause approximately 2 million deaths per year worldwide and had an increasing incidence during the last decade. Risk factors for liver diseases include alcohol consumption, obesity, diabetes, the intake of hepatotoxic substances like aflatoxin, viral infection, and genetic determinants. Liver cancer is the sixth most prevalent cancer and the third in mortality (second in males). The low survival rate (less than 20% in 5 years) is partially explained by the late diagnosis, which remarks the need for new early molecular biomarkers. One-carbon metabolism integrates folate and methionine cycles and participates in essential cell processes such as redox homeostasis maintenance and the regulation of methylation reactions through the production of intermediate metabolites such as cysteine and S-Adenosylmethionine. One-carbon metabolism has a tissue specific configuration, and in the liver, the participating enzymes are abundantly expressed-a requirement to maintain hepatocyte differentiation. Targeted proteomics studies have revealed significant differences in hepatocellular carcinoma and cirrhosis, suggesting that monitoring one-carbon metabolism enzymes can be useful for stratification of liver disease patients and to develop precision medicine strategies for their clinical management. Here, reprogramming of one-carbon metabolism in liver diseases is described and the role of mass spectrometry to follow-up these alterations is discussed.
    Keywords:  hepatocellular carcinoma; liver disease; one-carbon metabolism; proteomics
  59. Methods Mol Biol. 2022 ;2546 13-25
      Quality assurance (QA) activities enable continuous improvement through ongoing post-implementation monitoring to identify, evaluate, and correct problems. QA for clinical liquid chromatography tandem mass spectrometry (LC-MS/MS) assays should include specific components that address the unique aspects of these methods. This chapter briefly describes approaches for clinical LC-MS/MS system performance monitoring using batch and peak review metrics, largely following CLSI-C62A guidance. Though routine checks ensure the quality of results reported for each run, there is also a need to evaluate metrics between runs over time. Post-implementation performance monitoring of LC-MS/MS methods is typically focused on calibration curves, retention times, peak intensities, and ion ratios.
    Keywords:  Data analytics; LC-MS/MS; Mass spectrometry; Quality assurance
  60. Methods Mol Biol. 2022 ;2546 1-12
      Mass spectrometry is a technique that identifies analytes based on mass-to-charge (m/z) ratio and structural fragments. Although this technique has been used in research and specialized clinical laboratories for decades, only in recent years has mass spectrometry become popular in routine clinical laboratories. Mass spectrometry, especially when coupled with gas chromatography or liquid chromatography, provides very specific and often sensitive analysis of many analytes. Other advantages of mass spectrometry include simultaneous analysis of multiple analytes (>100) and generally limited requirement for specialized reagents. Commonly measured analytes by mass spectrometry include metabolites, drugs, hormones, and proteins.
    Keywords:  Clinical laboratory; Endocrinology; Gas chromatography; Hormones and proteins; Liquid chromatography; Mass spectrometry; Newborn screening; Tandem mass spectrometry
  61. J Lipid Res. 2022 Sep 14. pii: S0022-2275(22)00107-9. [Epub ahead of print] 100274
      Lipid accumulation in non-adipose tissues can cause lipotoxicity, leading to cell death and severe organ dysfunction. Adipose triglyceride lipase (ATGL) deficiency causes human Neutral Lipid Storage Disease and leads to cardiomyopathy; ATGL deficiency has no current treatment. One possible approach to alleviate this disorder has been to alter the diet and reduce the supply of dietary lipids and, hence, myocardial lipid uptake. However, in this study, when we supplied cardiac Atgl knockout mice a low- or high-fat diet, we found heart lipid accumulation, heart dysfunction, and death were not altered. We next deleted lipid uptake pathways in the ATGL-deficient mice through the generation of double knockout mice also deficient in either cardiac lipoprotein lipase (LpL) or cluster of differentiation (CD) 36, which is involved in an LpL-independent pathway for fatty acid uptake in the heart. We show neither deletion ameliorated ATGL-deficient heart dysfunction. Similarly, we determined non-lipid-containing media did not prevent lipid accumulation by cultured myocytes; rather, the cells switched to increased de novo fatty acid synthesis. Thus, we conclude pathological storage of lipids in ATGL deficiency cannot be corrected by reducing heart lipid uptake.
    Keywords:  CD36; LpL; dietary fat; fatty acid synthesis; heart failure; lipid accumulation; lipid droplets; lipotoxicity; myocardial lipid uptake; storage diseases
  62. Methods Mol Biol. 2023 ;2571 33-43
      Due to the high impact of diet exposure on health, it is crucial the generation of robust data of regular dietary intake, hence improving the accuracy of dietary assessment. The metabolites derived from individual food or group of food have great potential to become biomarkers of food intake (BFIs) and provide more objective food consumption measurements.Herein, it is presented an untargeted metabolomic workflow for the discovery BFIs in blood and urine samples, from the study design to the biomarker identification. Samples are analyzed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). A wide variety of compounds are covered by separate analyses of medium to nonpolar molecules and polar metabolites based on two LC separations as well as both positive and negative electrospray ionization. The main steps of data treatment of the comprehensive data sets and statistical analysis are described, as well as the principal considerations for the BFI identification.
    Keywords:  Biomarkers of food intake; Dietary assessment; Ion mobility; LC-HRMS; Plasma metabolites; Untargeted metabolomics; Urinary metabolites
  63. Methods Mol Biol. 2022 ;2546 261-269
      Deficiencies of the enzymes in lysosomes result in the accumulation of undegraded materials and subsequently cellular dysfunction. Early identification of deficiencies can lead to better clinical outcomes before irreversible organ and tissue damages occur. In this chapter, lysosomal enzymes are extracted from dried blood spots and incubated with the commercialized and multiplexed enzyme cocktail containing corresponding substrates and internal standards. After incubation, the enzymatic reactions are quenched, and the mixtures of the reaction products are prepared using liquid/liquid extractions. Multiple enzymes are quantified simultaneously using selected ion monitoring on liquid chromatography-mass spectrometry (LC-MS/MS) system.
    Keywords:  Dried blood spot; Enzyme activity assay; Liquid chromatography–mass spectrometry; Lysosomal storage disorders; Newborn screening
  64. Methods Mol Biol. 2022 ;2546 149-163
      Quantitation of long-chain fatty acids in serum/plasma and red blood cells is a useful diagnostic tool in the evaluation of nutritional status and assessment of risk for essential fatty acid deficiency (EFAD). Serum/plasma has been the traditional sample type for this method, yet it requires prolonged fasting which is not compatible with some patient populations. More recently, red blood cells have become an important sample type due to less intraindividual variability and obviating the need for fasting. Here we present a method for the quantitation of 22 fatty acids in serum/plasma or red blood cells. Fatty acids are hydrolyzed and extracted from the biological matrix, followed by derivatization with pentafluorobenzyl bromide and subsequent analysis by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS).
    Keywords:  Chemical ionization; Essential fatty acid deficiency; Fatty acids; Gas chromatography; Mass spectrometry; Plasma; Red blood cells; Selected ion monitoring; Serum
  65. Methods Mol Biol. 2022 ;2546 335-350
      A patient suspected of an inborn error of metabolism will commonly have urine organic acid analysis performed as part of their workup. The traditional urine organic acid method involves extraction of the acidic fraction from urine samples using an organic solvent, derivatization of extracted compounds, and identification using gas chromatography-mass spectrometry (GC/MS). Unfortunately, the extraction step results in the loss of many neutral and positively charged compounds which may be of interest to metabolic physicians and biochemical geneticists. By replacing the traditional extraction step with an enzymatic treatment of the sample with urease, an abundance of organic molecules is available for separation and quantification by GC/MS. The urease method is a useful adjunct to newborn screening follow-up, and it has the additional benefit of being able to identify many classes of biochemical compounds, such as amino acids, acylglycines, neurotransmitters, and carbohydrates. This method describes the urease treatment, derivatization, and the organic acids and other biochemical metabolites that can be identified.
    Keywords:  Gas chromatography–mass spectrometry; Inborn errors of metabolism; Organic acids; Urease
  66. Methods Mol Biol. 2023 ;2571 143-148
      Cholesterol is an essential lipid molecule for several biological functions including the proper functioning of cell membranes, lipoproteins, and lipid rafts, as well as the synthesis of bile acids, vitamin D, and steroid hormones. Cholesterol can be extracted from liver tissue by multiple methods of lipid extraction. Subsequently, gas chromatography-mass spectrometry (GC-MS) can be used to obtain the highest level of sensitivity and selectivity in the analysis of cholesterol. This chapter describes two methods of lipid extraction for liver tissue, Bligh and Dyer and methyl tertiary butyl ether (MTBE), followed by an analysis with GC-MS.
    Keywords:  Bligh and Dyer; Cholesterol; GC; GC–MS; Gas chromatography; Lipid extraction; Liver; MS; MTBE; Mass spectrometry
  67. Methods Mol Biol. 2022 ;2546 321-333
      Organic acidurias or acidemias are a group of diverse disorders caused by decreased or diminished activity of specific enzyme or transporter involved in the metabolism of amino acids, carbohydrates, fatty acids, and nucleic acids. Organic acidurias are generally inherited but may be acquired due to deficiency of certain cofactors or vitamins. As clinical symptoms are of nonspecific nature, definitive diagnosis of organic aciduria requires measurement of organic acids in urine or blood and sometimes enzyme activity in the cells. Gas chromatography-mass spectrometry (GC-MS) is a commonly used method for screening of organic acidurias.GC-MS procedure described here involves the use of urine volume that contains 1 μmole (113 μg) of creatinine. Internal standards (tropic and 2-ketocaproic acids) are added to the samples, followed by treatment with hydroxylamine to form oxime derivatives of the ketoacids. The mixture is then acidified, and organic acids are extracted in ethyl acetate. The organic extract is concentrated to dryness, and the residue is treated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS)/pyridine to form the trimethylsilyl (TMS) derivatives of the organic acids. The derivatized extract is then directly injected onto GC-MS for analysis.
    Keywords:  Gas chromatography–mass spectrometry; Organic acidemias; Organic acidurias; Trimethylsilyl derivatization