A sample extraction and chromatographic strategy for increasing LC/MS detection coverage of the erythrocyte metabolome

Sana, Theodore R.; Waddell, Keith; Fischer, Steven M.

doi:10.1016/j.jchromb.2008.04.030

Cited by 78 publications

(60 citation statements)

References 35 publications

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“…35,36 Finally, sample preparation (ie, RBC isolation and metabolite extraction) is a crucial step for metabolomics. Some described protocols rely on the use of thermal shocks 37 or sonication 38 for cell membrane disruption, followed by a metabolite extraction step using organic solvents. Compared with these methods, boiling RBCs at 100°C for 3 minutes in water followed by differential centrifugations gave the most reliable results in terms of number of metabolite detected and signal intensities.…”

Section: Discussionmentioning

confidence: 99%

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

Darghouth

Koehl

Madalinski³

et al. 2011

Blood

106

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Emerging metabolomic tools can now be used to establish metabolic signatures of specialized circulating hematopoietic cells in physiologic or pathologic conditions and in human hematologic diseases. To determine metabolomes of normal and sickle cell erythrocytes, we used an extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometrybased metabolite profiling method. Comparison of these 2 metabolomes identified major changes in metabolites produced by (1) endogenous glycolysis characterized by accumulation of many glycolytic intermediates; (2) endogenous glutathione and ascorbate metabolisms characterized by accumulation of ascorbate metabolism intermediates, such as diketogulonic acid and decreased levels of both glutathione and glutathione disulfide; (3) membrane turnover, such as carnitine, or membrane transport characteristics, such as amino acids; and (4) exogenous arginine and NO metabolisms, such as spermine, spermidine, or citrulline. Finally, metabolomic analysis of young and old normal red blood cells indicates metabolites whose levels are directly related to sickle cell disease. These results show the relevance of metabolic profiling for the follow-up of sickle cell patients or other red blood cell diseases and pinpoint the importance of metabolomics to further depict the pathophysiology of human hematologic diseases. (Blood. 2011; 117(6):e57-e66) IntroductionMetabolome is defined as the complete set of metabolites present in a given biologic system that can be unicellular organism, organ, tissue, cell, or biologically relevant liquid compartments. Complementary to genomics or proteomics, the aim of metabolome analysis is to describe qualitatively and quantitatively the final products of cellular regulatory pathways and can be seen as the ultimate response of a biologic system to genetic factors and/or environmental changes. 1 Presently, metabolomics is used to describe metabolites present in simple unicellular organisms, such as Escherichia coli, 2 or in important biologic fluids, such as plasma or urine, 3,4 but the cross-talks between cells in tissues or the complex metabolism in mammalian cells make the interpretation of metabolomics data challenging in human, although important metabolites involved in pathogenesis of cancer, 5,6 diabetes, 7 and cardiovascular 8,9 and mitochondrial diseases 10 are described.Mammalian mature red blood cell (RBC) is a cell without nucleus or cytoplasmic organelles, such as mitochondria or ribosomes, easy to collect and to purify in large quantity. In human, during their 120-day life span, RBCs are perfectly adapted to oxygen, carbon dioxide, and proton transport. RBCs have also an important role in interorgan transport of metabolites, such as amino acid transport to muscles resulting from numerous amino acids receptors on RBC membranes. RBCs are also used as reporters of exogenous metabolisms as exemplified by the level of hemoglobin A1c, which is a measure of erythrocyte hemoglobin glycation and reflects mean glycemia for the...

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Section: Discussionmentioning

confidence: 99%

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

Darghouth

Koehl

Madalinski³

et al. 2011

Blood

106

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“…Obviously, some analytes are ionized more efficiently in one ionization mode or one polarity and some in another mode. Currently, ESI is by far the method of choice in LC-MS metabolomic studies because it produces large numbers of ions via charge exchange in solution (Sana, Waddell, & Fischer, 2008). Atmospheric pressure ionization chemical ionization (APCI) on the other hand has been used in only a few cases.…”

Section: E Mass Spectrometry Platformsmentioning

confidence: 99%

“…A dual ionization metabolomics approach has recently been described increasing the coverage of the metabolome of human serum (Nordstrom et al, 2008). In another study a group from Agilent Technologies (Sana, Waddell, & Fischer, 2008) found that employing APCI in addition to ESI resulted in a 34% increase in the coverage (number of features detected) of the metabolome of human erythrocytes. Such effects are illustrated in Figure 3 (Sana, Waddell, & Fischer, 2008), which provides an interesting perspective on the differences between the ionization modes.…”

Section: E Mass Spectrometry Platformsmentioning

confidence: 99%

“…In another study a group from Agilent Technologies (Sana, Waddell, & Fischer, 2008) found that employing APCI in addition to ESI resulted in a 34% increase in the coverage (number of features detected) of the metabolome of human erythrocytes. Such effects are illustrated in Figure 3 (Sana, Waddell, & Fischer, 2008), which provides an interesting perspective on the differences between the ionization modes. It is well known that two mechanisms of ionization have some ion formation overlap (as also with APPI).…”

Section: E Mass Spectrometry Platformsmentioning

confidence: 99%

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Mass spectrometry‐based holistic analytical approaches for metabolite profiling in systems biology studies

Theodoridis

Gika

Wilson

2011

Mass Spectrometry Reviews

190

125

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Metabonomics and metabolomics represent one of the three major platforms in systems biology. To perform metabolomics it is necessary to generate comprehensive "global" metabolite profiles from complex samples, for example, biological fluids or tissue extracts. Analytical technologies based on mass spectrometry (MS), and in particular on liquid chromatography-MS (LC-MS), have become a major tool providing a significant source of global metabolite profiling data. In the present review we describe and compare the utility of the different analytical strategies and technologies used for MS-based metabolomics with a particular focus on LC-MS. Both the advantages offered by the technology and also the challenges and limitations that need to be addressed for the successful application of LC-MS in metabolite analysis are described. Data treatment and approaches resulting in the detection and identification of biomarkers are considered. Special emphasis is given to validation issues, instrument stability, and QA/quality control (QC) procedures.

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“…In recent years the development of new methods has seen metabolomics progress from a novel analytical technique towards a mainstay of the biological toolbox. However, before this transformation can be completed a number of technical challenges remain to be overcome, foremost among these is maximizing coverage of the metabolome [3,4]. A number of previous studies have done this by utilizing multiple complimentary techniques including liquid chromatography -mass spectrometry (LC-MS), nuclear magnetic resonance spectroscopy (NMR) and gas chromatography -mass spectrometry (GC-MS) [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Multimodal Metabolomics Combining UPLC-qToF-MS and GC-MS Data in Plasma and Brain Tissue

Ebshiana¹,

Snowden²,

Legido‐Quigley³

2017

Preprint

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Metabolomic analysis of biological fluids and tissues has become an increasingly routine tool in the biological toolbox. However, challenges remain to be overcome, including developing strategies to maximise coverage of the metabolome without requiring large sample volumes. Here we describe a multimodal strategy that combines data using both LC-MS and GC-MS from a unique vial with a sample of plasma (20µl) or a sample of brain tissue (3mg). Using a split phase extraction the non-aqueous phase was analyzed by reversed phase (RP) LC-MS, whilst the aqueous phase was analyzed using hydrophilic liquid interaction chromatography (HILIC)LC-MS, with both phases also analysed using GC-MS after derivatization of the extract. Analytical performance was assessed in 7 rat cerebellum samples and a pilot study of 40 plasma samples (20 vs. 20: AD vs. healthy controls). The method, which uses four hours of instrument time, measured 20,707 metabolite features in brain samples and 17,266 in plasma samples, from those 44.1% features displayed CV's below 15% and 75.2% below 30%. The method has potential to resolve subtle biological differences and to correlate metabolite composition directly to clinical outcomes including MMSE, age and ADCS-ADL. This method can acquire in the order of 20K metabolic features when low volumes are available.

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A sample extraction and chromatographic strategy for increasing LC/MS detection coverage of the erythrocyte metabolome

Cited by 78 publications

References 35 publications

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

Mass spectrometry‐based holistic analytical approaches for metabolite profiling in systems biology studies

Multimodal Metabolomics Combining UPLC-qToF-MS and GC-MS Data in Plasma and Brain Tissue

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