TITLE: Liquid chromatography-mass spectrometry method for isomer separation and detection of sugars, phophorylated sugars and organic acids
AUTHORS: Somnath  Koley, Kevin L. Chu, Saba S. Gill, Doug K. Allen

[DESCRIPTION]
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.

[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".

[STEPS]
SECTION: Abbreviations:
1. 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
SECTION: Apparatus
2. Vivaclear Mini   PES clarifying filters (Sartorius #VK01P042)
Durapore membrane filter, PVDF hydrophilic,  ,   (EMD Millipore #GVWP04700)
HPLC pump: Shimadzu Prominence-xR UFLC system 
HPLC column: Intrada Organic Acid (  x  ,  ) 
MS detector: Sciex QTRAP 6500 triple quadrupole-linear ion trap MS with a Turbo VTM electrospray ionization source
SECTION: HPLC conditions
5. Solvent A: ddH2O containing 100 millimolar (mM) ammonium formate and 10 % (v/v) acetonitrile 
Solvent B: ddH2O containing 1 % (v/v)  formic acid and 75 % (v/v) acetonitrile
Seal wash: ddH2O containing 20 % (v/v)  methanol and 0.5 % (v/v)  formic acid
Autosampler wash 1: ddH2O containing 25 % (v/v)  methanol
Autosampler wash 2: ddH2O containing 75 % (v/v)  methanol
Vacuum filter buffers using   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 50 % (v/v)  methanol with 0.2 % (v/v)  formic acid.
A 3 µL injection volume is used, the sample tray in autosampler is held at 4 °C, and the column temperature is held at 40 °C.
 
 


SECTION: LC-MS conditions (specific for QTRAP 6500 triple quadrupole MS)
6. Source temperature: 450 °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.
 
 
 

SECTION: Preparation of 100 μg/mL PIPES-Ribitol-Norvaline internal standards for sample extraction
4. 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).
SECTION: Preparing metabolite mixes for an external standard curve
3. 
SECTION: Preparing metabolite mixes for an external standard curve
3.1. A 500 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 0.2 % (v/v)  formic acid.

SECTION: Preparing metabolite mixes for an external standard curve
3.2. Prepare a  250 micromolar (µM) working stock in ddH2O containing  50 % (v/v)  methanol and  0.2 % (v/v)  formic acid.
SECTION: Preparing metabolite mixes for an external standard curve
3.3. Filter the  250 micromolar (µM) stock through a   PES clarifying filter at 2000 x g, 4 °C for 5 min.

SECTION: Preparing metabolite mixes for an external standard curve
3.4. Serially dilute the filtered  250 micromolar (µM) stock in a filtered ddH2O containing 50 % (v/v) methanol and  0.2 % (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.
SECTION: Preparing metabolite mixes for an external standard curve
3.5. Transfer 50 µL of each standard concentration into separate 300 µL LC vials with inserts.

SECTION: Determining sample metabolite concentrations from an external standard curve
7. 
SECTION: Determining sample metabolite concentrations from an external standard curve
7.1. 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.
SECTION: Determining sample metabolite concentrations from an external standard curve
7.2. 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.
SECTION: Determining sample metabolite concentrations from an external standard curve
7.3. In Excel, convert peak area units from µM to µmols of compound by multiplying by sample volume (50 µL).

SECTION: Determining sample metabolite concentrations from an external standard curve
7.4. Plot the peak areas vs the standard concentrations and fit a linear regression to the data.
SECTION: Determining sample metabolite concentrations from an external standard curve
7.5. 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.
SECTION: Determining sample metabolite concentrations from an external standard curve
7.6. For each metabolite, correct for metabolite loss during extraction using the relevant factor.
SECTION: Determining sample metabolite concentrations from an external standard curve
7.7. For each metabolite, calculate the concentration from the sample by solving for x using the linear regression equation of that metabolite’s standard curve.
SECTION: Calculation of limits of detection (LOD) and limits of quantitation (LOQ)
8. 
SECTION: Calculation of limits of detection (LOD) and limits of quantitation (LOQ)
8.1. 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.
SECTION: Calculation of limits of detection (LOD) and limits of quantitation (LOQ)
8.2. 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.

 
  
 




SECTION: Interpreting 13C-labeling in metabolites
9. 
SECTION: Interpreting 13C-labeling in metabolites
9.1. 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   

 
 
SECTION: Interpreting 13C-labeling in metabolites
9.2. Once runs are completed, extract the peak intensities (or areas) of the set of isotopologues for each compound.
SECTION: Interpreting 13C-labeling in metabolites
9.3. 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.

   
SECTION: Interpreting 13C-labeling in metabolites
9.4. 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.

    

SECTION: Preparation of 100 μg/mL PIPES-Ribitol-Norvaline internal standards for sample extraction
4.1. Prepare separate 1 mg/mL stocks for each internal standard in 1 mL chilled ddH2O.

SECTION: Preparation of 100 μg/mL PIPES-Ribitol-Norvaline internal standards for sample extraction
4.2. For 1 mL of 100 μg/mL standard mix, mix 100 µL of each 1 mg/mL stock with 700 µL chilled ddH2O.
SECTION: Preparation of 100 μg/mL PIPES-Ribitol-Norvaline internal standards for sample extraction
4.3. Add 15 µL Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants. 100 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 50 µL  ddH2O containing  50 % (v/v) methanol and  0.2 % (v/v)  formic acid and subsequently filtered through   PES clarifying filters at 2000 x g, 4 °C for 5 min.