Jan 12, 2023

Public workspaceLiquid chromatography-mass spectrometry method for isomer separation and detection of sugars, phophorylated sugars and organic acids

DOI
dx.doi.org/10.17504/protocols.io.b3mvqk66
  • 1Donald Danforth Plant Science Center;
  • 2United States Department of Agriculture-Agriculture Research Service, Donald Danforth Plant Science Center
Somnath Koley
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DOI: dx.doi.org/10.17504/protocols.io.b3mvqk66
Protocol Citation: Somnath Koley, Kevin L. Chu, Saba S. Gill, Doug K. Allen 2023. Liquid chromatography-mass spectrometry method for isomer separation and detection of sugars, phophorylated sugars and organic acids. protocols.io https://dx.doi.org/10.17504/protocols.io.b3mvqk66
Manuscript citation:
Koley, Somnath, et al. "An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids."Journal of experimental botany73.9 (2022): 2938-2952.
https://academic.oup.com/jxb/article/73/9/2938/6530308
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 10, 2022
Last Modified: January 12, 2023
Protocol Integer ID: 56725
Keywords: Central metabolism, isomer separation, isotopic labeling, liquid chromatography-mass spectrometry, metabolite quantification, mixed-mode column chromatography
Funders Acknowledgements:
National Science Foundation
Grant ID: DBI-1427621
National Science Foundation
Grant ID: IOS-1829365
USDA-National Institute of Food and Agriculture
Grant ID: 2017-67013-26156
Bill & Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the U.K. Foreign, Commonwealth and Development Office
Grant ID: OPP1172157
Abstract
This standard operating procedure is used to achieve effective separation of a wide range of polar metabolites found in central carbon metabolism via a hybrid liquid chromatographic method (ion-exchange chromatography and hydrophilic interaction liquid chromatography (HILIC)) using an Intrada Organic Acid column (Imtakt) coupled with triple quadrupole mass spectrometry. This method gives improved resolution while showing enhanced sensitivity for the detection of low abundance phosphorylated sugars compared with standard HILIC methods.
Materials
Reagents
  • Reagentdouble distilled water (ddH2O)
  • ReagentFormic acid (88%)Sigma AldrichCatalog #399388
  • ReagentMethanol (LCMS-grade)HoneywellCatalog #14262
  • ReagentAcetonitrile (UV/HPLC-grade)HoneywellCatalog #34888
  • ReagentAmmonium formateSigma AldrichCatalog #70221-25G-F
  • Metabolite standards (Sigma-Aldrich)







Before start
This protocol is part of the submitted paper "An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids".
Abbreviations:
Abbreviations:
  • CE: collision energy
  • CXP: collision cell exit potential
  • ddH2O: double-distilled water
  • DP: declustering potential
  • EP: entrance potential
  • FW: fresh weight
  • HILIC: hydrophilic interaction liquid chromatography
  • HPLC: high-performance liquid chromatography
  • ID: isotopologue distribution
  • LC-MS: liquid chromatography-mass spectrometry
  • MRM: multiple reaction monitoring
  • PES: Polyethersulfone
  • PIPES: Piperazine-N,N′-bis(2-ethanesulfonic acid)
  • PVDF: Polyvinylidene fluoride
Apparatus
Apparatus
  • Vivaclear Mini Thikness0.8 µm PES clarifying filters (Sartorius #VK01P042)
  • Durapore membrane filter, PVDF hydrophilic, Thikness0.22 µm , Thikness47 mm (EMD Millipore #GVWP04700)
  • HPLC pump: Shimadzu Prominence-xR UFLC system
  • HPLC column: Intrada Organic Acid (Thikness150 mm x Thikness2 mm , Thikness3 µm )
  • MS detector: Sciex QTRAP 6500 triple quadrupole-linear ion trap MS with a Turbo VTM electrospray ionization source
Preparing metabolite mixes for an external standard curve
Preparing metabolite mixes for an external standard curve

A Concentration500 micromolar (µM) stock of alpha-ketoglutarate, 2-phosphoglycolate, 2-phosphoglyceric acid, 3-phosphoglyceric acid, 6-phosphogluconate, acetyl-CoA, adenosine diphosphate-glucose, aspartate, dihydroxyacetone phosphate, erythrose 4-phosphate, fructose, fructose 1,6-bisphosphate, glucose 1-phosphate, glucose 6-phosphate, glyceraldehyde phosphate, glucose, glutamine, glutamate, glycerate, malate, phosphoenolpyruvate, piperazine-N,N′-bis(2-ethanesulfonic acid), raffinose, ribitol, ribulose-1,5-bisphosphate, ribulose 5-phosphate, sedoheptulose 7-phosphate, succinate, sucrose, and uridine diphosphate-glucose standards was prepared in ddH2O containing Concentration0.2 % (v/v) formic acid.

Prepare a Concentration250 micromolar (µM) working stock in ddH2O containing Concentration50 % (v/v) methanol and Concentration0.2 % (v/v) formic acid.
Filter the Concentration250 micromolar (µM) stock through a Thikness0.8 µm PES clarifying filter at Centrifigation2000 x g, 4°C for Duration00:05:00 .

5m
Serially dilute the filtered Concentration250 micromolar (µM) stock in a filtered ddH2O containing Concentration50 % (v/v) methanol and Concentration0.2 % (v/v) formic acid to create a standard curve containing 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, 0.977, 0.488, 0.244, and 0.122 µM standards.
Transfer Amount50 µL of each standard concentration into separate Amount300 µL LC vials with inserts.

Preparation of 100 μg/mL PIPES-Ribitol-Norvaline internal standards for sample extraction
Preparation of 100 μg/mL PIPES-Ribitol-Norvaline internal standards for sample extraction
These internal standards are used for calculation of extraction efficiencies (PIPES for organic acids, ribitol for sugars and phosphorylated sugars, and norvaline for amino acids).
Prepare separate Concentration1 mg/mL stocks for each internal standard in Amount1 mL chilled ddH2O.

For Amount1 mL of Concentration100 μg/mL standard mix, mix Amount100 µL of each Concentration1 mg/mL stock with Amount700 µL chilled ddH2O.
Add Amount15 µL Concentration100 μg/mL PIPES-Ribitol-Norvaline as internal standards during sample extraction. Polar metabolites are extracted using the protocol adapted from Ma et al., 2017, with only a single water extraction step performed and samples being resuspended in Amount50 µL ddH2O containing Concentration50 % (v/v) methanol and Concentration0.2 % (v/v) formic acid and subsequently filtered through Thikness0.8 µm PES clarifying filters at Centrifigation2000 x g, 4°C for Duration00:05:00 .
CITATION
Ma F, Jazmin LJ, Young JD, Allen DK (2017). Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants.. Methods in molecular biology (Clifton, N.J.).
LINK
https://doi.org/10.1007/978-1-4939-7225-8_12


5m
HPLC conditions
HPLC conditions
  • Solvent A: ddH2O containing Concentration100 millimolar (mM) ammonium formate and Concentration10 % (v/v) acetonitrile
  • Solvent B: ddH2O containing Concentration1 % (v/v) formic acid and Concentration75 % (v/v) acetonitrile
  • Seal wash: ddH2O containing Concentration20 % (v/v) methanol and Concentration0.5 % (v/v) formic acid
  • Autosampler wash 1: ddH2O containing Concentration25 % (v/v) methanol
  • Autosampler wash 2: ddH2O containing Concentration75 % (v/v) methanol
  • Vacuum filter buffers using Thikness0.22 µm PVDF hydrophilic membrane filters into clean bottles.

Table 1. HPLC mobile phase gradient
Time (min) Flow (mL/min) %A %B
0 0.225 0 100
1 0.225 0 100
5 0.225 12 88
7 0.225 12 88
8 0.225 16 84
10 0.225 16 84
13 0.225 25 75
15 0.225 100 0
19.5 0.25 100 0
20 0.25 0 100
25 0.25 0 100
  • All samples are diluted in ddH2O containing Concentration50 % (v/v) methanol with Concentration0.2 % (v/v) formic acid.
  • A Amount3 µL injection volume is used, the sample tray in autosampler is held at Temperature4 °C , and the column temperature is held at Temperature40 °C .


LC-MS conditions (specific for QTRAP 6500 triple quadrupole MS)
LC-MS conditions (specific for QTRAP 6500 triple quadrupole MS)
  • Source temperature: Temperature450 °C
  • Ion spray voltage: -4500 V
  • Curtain gas: 30 psi
  • Ion source gas 1: 30 psi
  • Ion source gas 2: 35 psi

Table 2. LC-MS parameters for each compound
Q1 Q3 Compound ID DP (v) CE (v) CXP (v) EP (v)
145 101 Alpha-ketoglutarate -5 -12 -11 -10
155 79 2-phosphoglycolate -70 -20 -12 -10
275 79 6-phosphogluconate -65 -22 -13 -10
808 408 Acetyl-CoA -35 -46 -23 -10
588 346.1 Adenosine diphosphate-glucose -30 -32 -21 -10
132 88 Aspartate -20 -16 -43 -10
199 97 Erythrose 4-phosphate -5 -32 -11 -10
339 97 Fructose 1,6-bisphosphate -60 -24 -11 -10
145.1 108.9 Glutamine -30 -18 -5 -10
146.05 102 Glutamate -16 -15 -8 -10
179 89 Hexoses -25 -10 -11 -10
259 79 Hexose phosphates -10 -28 -7 -10
133 115 Malate -40 -20 -3 -10
229 79 Pentose 5-phosphates -25 -60 -15 -10
167 79 Phosphoenolpyruvate -5 -14 -9 -10
185 79 Phosphoglyceric acids -30 -20 -11 -10
301 193 PIPES -35 -34 -31 -10
87 43 Pyruvate -10 -15 -3 -10
151 89 Ribitol -50 -16 -9 -10
309 79 Ribulose 1,5-bisphosphate -55 -70 -9 -10
289 79 Sedoheptulose 7-phosphate -60 -58 -9 -10
104.001 74 Serine -16 -15 -8 -10
117 73 Succinate -5 -16 -7 -10
341 179 Sucrose -110 -18 -13 -10
169 97 Triose phosphates -10 -12 -11 -10
565 323.1 Uridine diphosphate-glucose -125 -32 -2 -10
All analytes are measured in negative ionization mode, with ions being detected using a targeted MRM approach.

Determining sample metabolite concentrations from an external standard curve
Determining sample metabolite concentrations from an external standard curve

Run a full set of all 12 concentrations of the external standard mix on the LC-MS three times total, at the start, middle, and end of sample runs.
Integrate peak areas for each standard peak using the quantitation wizard tool in the Analyst instrument control and data processing software (v.1.6.2) and export the data to Excel.
In Excel, convert peak area units from µM to µmols of compound by multiplying by sample volume (Amount50 µL ).

Plot the peak areas vs the standard concentrations and fit a linear regression to the data.
For sample runs, compare the measured amounts of PIPES, ribitol, and norvaline internal standards with the known amounts added during sample preparation to calculate the percent metabolite recovery, using PIPES for organic acids, norvaline for amino acids, and ribitol for sugars, nucleotide sugars, and phosphorylated sugars.
For each metabolite, correct for metabolite loss during extraction using the relevant factor.
For each metabolite, calculate the concentration from the sample by solving for x using the linear regression equation of that metabolite’s standard curve.
Calculation of limits of detection (LOD) and limits of quantitation (LOQ)
Calculation of limits of detection (LOD) and limits of quantitation (LOQ)

For each metabolite, determine the lowest concentration on the standard curve that still showed a change in peak area. Calculate the standard deviation of this concentration (SDlow) for all three injections.
Calculate the limits of detection and quantitation for each metabolite by multiplying this standard deviation by 3 or 10 and dividing by the slope of the standard curve.






Interpreting 13C-labeling in metabolites
Interpreting 13C-labeling in metabolites

For sample runs investigating 13C-tracer incorporation, the MRM list for compounds of interest was expanded with the set of possible labeled carbon isotopologues in the Q1 ion and the Q3 fragment (if applicable). Since the Q3 fragment for a compound like 3-PGA does not contain any carbons, an increase in label is only possible in the Q1 ion.

Table 3. Monitored isotopologue distribution (ID) for select metabolites
Q1 Q3 Compound ID (13C in Q1/13C in Q3)
133 115 MAL-0/0
134 116 MAL-1/1
135 117 MAL-2/2
136 118 MAL-3/3
137 119 MAL-4/4
185 79 PGA-0/0
186 79 PGA-1/0
187 79 PGA-2/0
188 79 PGA-3/0

Once runs are completed, extract the peak intensities (or areas) of the set of isotopologues for each compound.
To determine isotopologue distribution (ID) of a particular isotopologue (Mi), divide the abundance (A) of that isotopologue by the sum of all isotopologue abundances for that compound.

with n being the number of carbons in that compound
Once all of the isotopologue distributions have been determined, the average 13C-enrichment for each compound can be calculated by determining the sum of each isotopologue multiplied by the number of labeled carbons present and dividing by total number of carbons in that compound.



Citations
Step 4.3
Ma F, Jazmin LJ, Young JD, Allen DK. Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants.
https://doi.org/10.1007/978-1-4939-7225-8_12