1 WATERS SOLUTIONS ACQUITY UPLC ® System ACQUITY UPLC BEH Amide Column SYNAPT ® G2 System TransOmics™ Informatics KEY WORDS HILIC, UPLC ® , small polar metabolites, QTof MS, metabolomics APPLICATION BENEFITS The combination of UPLC-based hydrophilic interaction liquid chromatography (HILIC) and a hybrid quadrupole time-of-flight (Q-Tof™) mass spectrometer allows the comprehensive analysis of small polar metabolites including sugars, phosphorylated compounds, purines and pyrimidines, nucleotides, nucleosides, acylcarnitines, organic acids, hydrophilic vitamins, and amino acids. Retention times and accurate masses of metabolites involved in key metabolic pathways were annotated for routine high-throughput screening in both untargeted and targeted metabolomics analyses. INTRODUCTION Metabolomics, a powerful tool in systems biology, aims to screen small metabolites present in biological samples. Differences in the species or amounts of metabolites can be used to characterize phenotypes and biological responses to perturbations (diseases, genetic modifications, or nutritional and pharmacological treatments). Small metabolites can be mainly divided into hydrophilic and hydrophobic compounds. Because water is the major constituent of cells, a high number of hydrophilic metabolites are present in their intracellular content including sugars, phosphorylated compounds, nucleobases, nucleotides, nucleosides, acylcarnitines, organic acids, hydrophilic vitamins, and amino acids, as shown in Figure 1. Such polar metabolites are the building blocks of large macromolecules such as nucleic acids (DNA and RNA), proteins, and oligosaccharides. Furthermore, they are involved in central pathways (glycolysis, pentose-phosphate pathway and citric acid cycle), which are essential for energy metabolism. Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC/QTof MS Giuseppe Paglia, 1 James Langridge, 2 and Giuseppe Astarita 3 Center for Systems Biology, University of Iceland, Iceland; 2-3 . Waters Corporation, Manchester, UK and Milford, MA, USA Organic acid: e.g., Succinic Acid Amino acids: e.g., L-Serine Sugar-phosphate: Fructose 6-phosphate Sugar: e.g., Sucrose Nucleotide: e.g., Adenosine-5'-triphosphate Nucleobase: e.g., Adenine NH 2 HO O OH Figure 1. Representative classes of polar metabolites present in biological samples.
10
Embed
Development of a Metabolomic Assay for the Analysis of Polar … · 2015. 7. 20. · Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC-TOF
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
WAT E R S SO LU T IO NS
ACQUITY UPLC® System
ACQUITY UPLC BEH Amide Column
SYNAPT® G2 System
TransOmics™ Informatics
K E Y W O R D S
HILIC, UPLC®, small polar metabolites,
QTof MS, metabolomics
A P P L I C AT IO N B E N E F I T S
The combination of UPLC-based hydrophilic
interaction liquid chromatography (HILIC) and
a hybrid quadrupole time-of-flight (Q-Tof™)
mass spectrometer allows the comprehensive
analysis of small polar metabolites including
sugars, phosphorylated compounds, purines
and pyrimidines, nucleotides, nucleosides,
acylcarnitines, organic acids, hydrophilic
vitamins, and amino acids. Retention times and
accurate masses of metabolites involved in key
metabolic pathways were annotated for routine
high-throughput screening in both untargeted
and targeted metabolomics analyses.
IN T RO DU C T IO N
Metabolomics, a powerful tool in systems biology, aims to screen small
metabolites present in biological samples. Differences in the species or
amounts of metabolites can be used to characterize phenotypes and biological
responses to perturbations (diseases, genetic modifications, or nutritional
and pharmacological treatments).
Small metabolites can be mainly divided into hydrophilic and hydrophobic
compounds. Because water is the major constituent of cells, a high number
of hydrophilic metabolites are present in their intracellular content including
acylcarnitines, organic acids, hydrophilic vitamins, and amino acids, as shown in
Figure 1. Such polar metabolites are the building blocks of large macromolecules
such as nucleic acids (DNA and RNA), proteins, and oligosaccharides. Furthermore,
they are involved in central pathways (glycolysis, pentose-phosphate pathway
and citric acid cycle), which are essential for energy metabolism.
Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC/QTof MSGiuseppe Paglia,1 James Langridge,2 and Giuseppe Astarita3
Center for Systems Biology, University of Iceland, Iceland;2-3. Waters Corporation, Manchester, UK and Milford, MA, USA
Figure 2. Representative UPLC/MS separation of selected polar metabolites using both basic and acidic chromatographic conditions.
5Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC-TOF
Figure 3. Basic chromatographic conditions allow for better separation of nucleoside phosphates compared to acidic conditions (Panels A and B); for method details please see Experimental section. Similarly, columns based on 150 mm HILIC amide chemistry improve the analysis of nucleoside phosphates compared to 150 mm reversed-phase HSS T3 C18 chemistry (Panel C; for method details please see reference 1).
In fact, the analysis of polar metabolites was strongly influenced by pH of the mobile phase, as shown in
Figures 3A and 3B. In particular, many phosphorylated compounds, such as nucleotides, could be well
separated using basic conditions; whereas, they were strongly retained using acidic conditions resulting
in poor chromatographic peak shape, as shown in Figures 3A and 3B. Notably, the analysis of similar
compounds using traditional reversed-phase LC/MS presented some challenges due to the fact that these
metabolites were poorly retained and usually eluted in the void volume, as shown in Figure 3C.1 Using HILIC
conditions, the set of polar metabolites eluted in order of increasing polarity. Retention times were annotated,
as well as with the accurate masses of precursor, adducts, and fragment ions using both ES+ and ES-,
as shown in Table 2.
2 341
1. FAD2. ADP-Glucose3. ADP-Ribose4. UDP-Glucose
1
3
4
2
BASIC ACIDIC
1
1. AMP2. ADP3. ATP
1
32
BASIC ACIDIC
A
B
C
6Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC-TOF
7Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC-TOF
Calibration curves were obtained for various chemical classes of the metabolites, shown in Figure 4, which
displayed a linear coefficient (Pearson’s correlation, R2) higher than 0.99. The LOD was lower than 100 ng/mL
for most of the analytes reported in Table 2.
Figure 4. Calibration curves from selected small polar metabolites.
8Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC-TOF
To test the applicability of this HILIC-UPLC/MS strategy in real biological samples, polar metabolites
extracted from human platelets were analyzed. Polar metabolites were separated with excellent retention
time reproducibility, shown in Figure 5, acquiring accurate mass information from m/z 50 to m/z 1000.
As general workflow, untargeted analyses were performed on this dataset using TransOmics Informatics
tools for the visualization, processing, and interpretation of MS data, allowing the discovery and identification
of unexpected alterations among sample groups (data not shown).
POSAcidic Condition
NEGBasic Condition
NEGAcidic Condition
Figure 5. Overlaid chromatographic trace of multiple injections of polar metabolites exctracted from platelets. Each polar metabolite extract was injected eight times during two different batches (80 injections) in two different days of analysis.
9Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC-TOF
Additionally, targeted analyses were conducted using the list of retention times and masses information
reported in Table 2, allowing the identification and quantification of the most common metabolites present
in biological samples, as shown in Figures 6A and 6B.
AMP
IMP
Inosine
UMP
GMP
Glucose-6-PFructose-6-P
Basic ES-
Arginine
SAH
Carnitine
5-MTA
Fragment Inosine
Fragment IMPHypoxanthine
A BAcid ES+
Figure 6. Representative UPLC/MS chromatograms of polar metabolites extracted from platelets and analyzed using acidic conditions for positive ES (A) and basic conditions for negative ES (B).
Waters Corporation34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters, ACQUITY UPLC, SYNAPT, and UPLC are registered trademarks of Waters Corporation. Q-Tof, TransOmics, and T he Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners