Oct 01, 2024

Public workspaceJGI/LBNL Metabolomics - Standard LC-MS/MS ESI Method - Polar HILIC-Z

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxydwkl8j/v1
  • Katherine Louie1,
  • Suzanne Kosina2,
  • Thomas Harwood1,
  • Meghana Faltane1,
  • Marie Lynde1,
  • Benjamin Bowen1,2,
  • Trent Northen1,2
  • 1Lawrence Berkeley National Laboratory, Joint Genome Institute, Berkeley, CA, United States;
  • 2Lawrence Berkeley National Laboratory, Environmental Genomics and Systems Biology Division, Berkeley, CA, United States
  • JGI/LBNL Metabolomics Repository
Katherine B. Louie
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxydwkl8j/v1
Protocol Citation: Katherine Louie, Suzanne Kosina, Thomas Harwood, Meghana Faltane, Marie Lynde, Benjamin Bowen, Trent Northen 2024. JGI/LBNL Metabolomics - Standard LC-MS/MS ESI Method - Polar HILIC-Z. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxydwkl8j/v1
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: September 26, 2024
Last Modified: October 01, 2024
Protocol Integer ID: 108480
Keywords: metabolomics, JGI, HILIC, LBNL, Joint Genome Institute, LC-MS, Thermo Orbitrap, Berkeley Lab, Environmental Metabolite Atlas, jgi metabolomic, lbnl metabolomic, metabolomic, mass spectrometry dataset, consistent outcomes across mass spectrometry dataset, confident annotation of metabolite, environmental metabolite atlas, thermo orbitrap mass spectrometer with esi source, polar metabolite, primary metabolite, metabolite, thermo orbitrap mass spectrometer, chromatography, ms esi method, using normal phase chromatography, normal phase chromatography, thousands of compound standard, fragmentation spectra
Funders Acknowledgements:
The work conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy
Grant ID: Contract No. DE-AC02-05CH11231
The work conducted by ENIGMA – Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research and used resources of the National Energy Research Scientific Computing Center, a Department of Energy Office of Science User Facility
Grant ID: Contract No. DE-AC02-05CH11231
Abstract
This protocol describes the standard LC-MS/MS ESI method developed at Lawrence Berkeley National Laboratory (LBNL) by JGI Metabolomics and EGSB's Northen Lab to analyze polar metabolites (e.g. amino acids, nucleic acids, sugars, organic acids, primary metabolites, etc.) using normal phase chromatography (HILIC-Z) coupled to a Thermo Orbitrap Mass Spectrometer with ESI source. This robust method of detection is easily reproduced and adapted onto similar LC-MS/MS systems to achieve consistent outcomes across mass spectrometry datasets, foster inter-lab and inter-experiment comparability, and enable effective data integration and analysis. Since its inception in 2018, this method has been used to analyze tens of thousands of experimental samples, with many of these datasets publicly available in the MassIVE data repository1,2. This method has also been used to run thousands of compound standards to collect characteristic retention time, m/z and fragmentation spectra. This forms the basis of Berkeley Lab's Environmental Metabolite Atlas (EMA), a database used for confident annotation of metabolites and publicly available through GNPS/GNPS23.
Troubleshooting
Overview
This protocol describes the standard LC-MS/MS ESI method developed at Lawrence Berkeley National Laboratory (LBNL) by JGI Metabolomics and EGSB's Northen Lab to analyze polar metabolites (e.g. amino acids, nucleic acids, sugars, organic acids, primary metabolites, etc.) using normal phase chromatography (HILIC-Z) coupled to a Thermo Orbitrap Mass Spectrometer with ESI source.

Instrumentation / Equipment
Mass Spectrometry Instrumentation

ABCD
Mass SpectrometerSource / ProbeESI needle (calibration)ESI needle (running samples)
Thermo Q Exactive HF Orbitrap (QE-HF)Thermo Ion Max API Source w/ H-ESI II probe32G Metal Needle High Flow (OPTON-53010, Thermo)32G Metal Needle High Flow (OPTON-53010, Thermo)
Thermo Q Exactive Orbitrap (QE)Thermo Ion Max API Source w/ H-ESI II probe32G Metal Needle High Flow (OPTON-53010, Thermo)32G Metal Needle High Flow (OPTON-53010, Thermo)
Thermo Orbitrap Exploris 120 (Exp120)Thermo OptaMax NG™ API source w/ H-ESI probe35G Metal Needle Low Flow, 50 um ID (OPTON-30139)32G Metal Needle High Flow, 100 um ID (OPTON-30694)
Thermo Orbitrap IQ-X Tribrid (IQX)Thermo OptaMax NG™ API source w/ H-ESI probe35G Metal Needle Low Flow, 50 um ID (OPTON-30139)32G Metal Needle High Flow, 100 um ID (OPTON-30694)
Table 1. Mass spectrometer and source.
Note: Other Thermo Orbitrap mass spectrometers may also be used (e.g. IDX, Exploris 240, Astral, etc) with appropriate source, ESI needle and modified acquisition parameters.
UHPLC System

ABCD
Module NameModule TypePart NumbersAlternative Part Numbers
1290 Infinity DAD*Diode Array DetectorG4212AG7115A, G7117BR
1200 Infinity Series TCCColumn CompartmentG1316AG7116B
1290 Infinity SamplerAutosamplerG4226AG7167B - Multisampler w/ thermostat
1290 Infinity ThermostatAutosampler ThermostatG1330BG7167B - Multisampler w/ thermostat
1290 Infinity Bin PumpBinary PumpG4220AG7120A
Table 2. Agilent 1290 Infinity UHPLC Modules and type.
* Optional

Note: To use Agilent LC systems in line with Thermo Orbitrap mass spectrometers, communication / compatibility requires either installation of Chromeleon software, or a contact closure board (for QE-HF or QE) or Universal Interface Box (UIB) (for IQX, IDX, and Exploris models), as well as specialized software packages and cables. Information, manuals and installation procedures are available from Agilent and Thermo.
UHPLC Column information

AB
Column nameInfinityLab Poroshell 120 HILIC-Z
Part ##683775-924
ManufacturerAgilent
Column chemistryHILIC-Z, zwitterionic
Inner Diameter (ID)2.1 mm
Length150 mm
Particle size2.7 µm
Pore size100 Å
Max pressure600 bar
pH range2-12
Max temperature80 ºC @ pH 7; 35 ºC @ pH 2-12
Table 3. UHPLC column information.

Chemicals / solvents
AB
Chemicals / solventsProduct Number
acetonitrile (LC-MS grade)AX0156, Sigma
water (LC-MS grade)9831-03, VWR
ammonium acetate (LC-MS grade) 73594, Sigma
acetic acid (glacial, ≥99.7%)JT9515-3, VWR
methylene-di-phosphonic acid (medronic acid)64255, Sigma
methanol (LC-MS grade)MX0486, Sigma
Table 4. Chemicals and solvents. These are used to prepare mobile phase and resuspend extracts. For solvents, other LC-MS grade products can also be used. For chemicals, high purity compounds of analytical grade or listed as suitable for mass spectrometry can be used.


LC-MS/MS Method Parameters
LIQUID CHROMATOGRAPHY

AB
Mobile Phase A99.8% H2O and 0.2% acetic acid, w/ 5 mM ammonium acetate and 5 µM methylene-di-phosphonic acid
Mobile Phase B99.8% 95:5 v/v ACN:H2O and 0.2% acetic acid, w/ 5 mM ammonium acetate
Table 5. Mobile phase composition for HILIC. Sufficient mobile phase for all injections of a sample set are prepared prior to starting a run. To prepare Mobile Phase B, ammonium acetate is first dissolved in water prior to adding acetonitrile and other components (for solubility).

Column
InfinityLab Poroshell 120 HILIC-Z, 2.1 × 150 mm, 2.7 µm, 100 Å (Agilent, #683775-924)

Column Temperature Temperature40 °C
Autosampler Temperature Temperature4 °C

ABCDE
Time (min)Flow (mL/min)%A%Bminutes for segment
00.450100
10.4501001
110.45118910
15.750.4530704.75
16.250.4580200.5
18.50.4580202.25
18.60.4501000.1
210.4501002.4
Table 6. Mobile phase gradients for HILIC. Each segment is a linear gradient to the new mobile phase composition.

Typically, depending on the length of the lines connecting the ESI needle through to the autosampler, the first 0.3-0.8 minutes of the run does not contain signal from the injected sample (void volume). This is then followed by a large peak (solvent front) comprised of metabolites that do not retain on the column. Signals acquired during this time window are typically not used in analysis. Also, signals acquired in the time window following final isocratic elution (here, between 18.5 to 21 minutes during column re-equilibration), are also not used in analysis.

Salty samples: When analyzing samples that still have a lot of salt in the extract, diverting flow to waste for the first 1 minute (void volume containing the most salt) may help keep the ESI needle and source cleaner throughout the run and preserve data quality.
MASS SPECTROMETRY

Source settings
ABCDE
ParameterQE-HFQEExp120IQX
Sheath Gas Flow Rate (au)55555050
Auxillary Gas Flow Rate (au)20201010
Sweep Gas Flow Rate (au)2211
Spray Voltage (V) - POS3000300035003500
Spray Voltage (V) - NEG3000300025002500
Capillary Temperature (°C)400400325325
Vaporizer Temperature (°C)N/AN/A300300
S-Lens RF Level (%)50507050
Table 7. ESI source settings. These settings are used with these specific Orbitrap models. Other mass spectrometers will need these source settings adjusted to achieve similar results.
  • au = arbitrary units

MS1 Settings
ABCDE
ParameterQE-HFQEExp120IQX
Microscans 1111
Resolution 60,00070,00060,00060,000
AGC Target3e63e6Standard (100%)1e5
Maximum IT (ms)100100Auto118
Scan range (m/z)70 - 1050 70 - 1050 70 - 1050 70 - 1050
Spectrum data typeCentroidCentroidCentroidCentroid
Table 8. MS1 scan settings. Full MS spectra are collected in both positive and negative ionization modes. These settings are used with these specific Orbitrap models. Other mass spectrometers will need these source settings adjusted to achieve similar results.

Collection time: 21 minutes

MS2 Settings
ABCDE
ParameterQE-HFQEExp120IQX
Microscans1111
Resolution 15,00017,50015,00015,000
AGC Target1e51e5Standard (100%)5e4
Maximum IT (ms)5050Auto22
Loop count2 (or 4)2 (or 4)4N/A
Cycle timeN/AN/AN/A0.8 sec
MSX count1111
TopN2 (or 4)2 (or 4)410-15
Exclusion duration (sec)7 - 107 - 1045
Stepped Collision Energies (eV)10, 20, 40 (or 20, 50, 60)10, 20, 40 (or 20, 50, 60)10, 20, 40 (or 20, 50, 60)10, 20, 40 (or 20, 50, 60)
Table 9. MS2 scan settings for collecting fragmentation data. Stepped and then averaged collision energies of 10, 20, 40 eV and/or 20, 50, 60 eV. A full MS1 scan is followed by "N" MS2 scans of the most intense precursor ions (TopN), excluding those precursors already fragmented in the previous time period (exclusion duration).

Source and acquisition settings listed here are the settings used for standard LC-MS/MS runs. Depending on experimental details and goals, these can be adjusted (e.g. instrument resolution, customization of data-dependent MS2, scan range, etc) as needed.

Sample Vial Preparation
Quality Control (QC) Mix
This is a custom formulation of compounds with annotated m/z, retention time (RT), and MS2 spectra dissolved in 100% MeOH. Compounds are representative of the metabolite classes detected using this LC-MS/MS method, as well as m/z and retention time ranges.

Usage: QC injections are interspersed throughout the LC-MS run to monitor instrument performance (calibration, intensity, retention time, etc.) as well as adjust compound retention times between runs.

ABCDEFGH
CompoundFormulaMonoisotopic massAdduct (POS)m/z (POS)Adduct (NEG)m/z (NEG)Expected RT
1-methyladenosineC11H15N5O4281.1124[M+H]+282.1197[M-H]-280.105110.78
2,4-dihydroxypteridineC6H4N4O2164.0334[M+H]+165.0407[M-H]-163.02611.27
2'-deoxyadenosineC10H13N5O3251.1018[M+H]+252.1091[M-H]-250.09462.23
2'-deoxyguanosineC10H13N5O4267.0968[M+H]+268.1040[M-H]-266.08956.87
2-hydroxyphenylacetic acidC8H8O3152.0473[M+H]+153.0546[M-H]-151.04011.62
2-oxovaleric acidC5H8O3116.0473[M+H]+117.0546[M-H]-115.04014.45
4-coumaric acidC9H8O3164.0473[M+H]+165.0546[M-H]-163.04011.51
4-guanidinobutanoic acidC5H11N3O2145.0851[M+H]+146.0924[M-H]-144.077913.86
4-methoxyphenylacetic acidC9H10O3166.0630[M+H]+167.0703[M-H]-165.05571.07
5-methylcytosineC5H7N3O125.0589[M+H]+126.0662[M-H]-124.05164.42
5-oxo-prolineC5H7NO3129.0426[M+H]+130.0499[M-H]-128.035311.65
ABMBA (2-amino-3-bromo-5-methylbenzoic acid)C8H8BrNO2228.9738[M+H]+229.9811[M-H]-227.96661.20
abscisic acidC15H20O4264.1362[M+H]+265.1434[M-H]-263.12891.15
acetylcholineC7NH16O2+146.1176[M]+146.1176[M-2H]-144.10301.96
adenineC5H5N5135.0545[M+H]+136.0618[M-H]-134.04722.56
adenosineC10H13N5O4267.0968[M+H]+268.1040[M-H]-266.08953.09
alanineC3H7NO289.0477[M+H]+90.0550[M-H]-88.040413.41
alpha-glucoseC6H12O6180.0634[M+H]+181.0707[M-2H]2-89.07075.06
alpha-ketoglutaric acidC5H6O5146.0215[M+H]+147.0288[M-H]-145.014214.51
arginineC6H14N4O2174.1117[M+H]+175.1190[M-H]-173.104416.94
asparagineC4H8N2O3132.0535[M+H]+133.0608[M-H]-131.046214.37
aspartic acidC4H7NO4133.0375[M+H]+134.0448[M-H]-132.030216.13
benzoic acidC7H6O2122.0368[M+H]+123.0441[M-H]-121.02951.27
betaineC5H12NO2+118.0863[M]+118.0863[M-2H]-116.07177.91
caffeic acidC9H8O4180.0423[M+H]+181.0495[M-H]-179.03503.22
caffeineC8H10N4O2194.0804[M+H]+195.0877[M-H]-193.07310.88
carnitineC7H16NO3+162.1125[M+H]+162.1125[M-H]-160.097913.29
cis-4-hydroxy-prolineC5H9NO3131.0582[M+H]+132.0655[M-H]-130.051013.67
citrullineC6H13N3O3175.0957[M+H]+176.1030[M-H]-174.088415.09
creatineC4H9N3O2131.0695[M+H]+132.0768[M-H]-130.062213.39
cysteic acidC3H7NO5S169.0045[M+H]+170.0118[M-H]-167.997214.54
cytidineC9H13N3O5243.0855[M+H]+244.0928[M-H]-242.07826.93
cytosineC4H5N3O111.0433[M+H]+112.0505[M-H]-110.03604.83
deoxycytidineC9H13N3O4227.0906[M+H]+228.0979[M-H]-226.08335.59
deoxyuridineC9H12N2O5228.0746[M+H]+229.0819[M-H]-227.06731.88
ectoineC6H10N2O2142.0742[M+H]+143.0815[M-H]-141.067012.50
fumaric acidC4H4O4116.0110[M+H]+117.0182[M-H]-115.003716.31
gamma-aminobutyric acidC4H9NO2103.0633[M+H]+104.0706[M-H]-102.056114.39
glutamic acidC5H9NO4147.0532[M+H]+148.0604[M-H]-146.045915.94
glutamineC5H10N2O3146.0691[M+H]+147.0764[M-H]-145.061914.31
glutaric acidC5H8O4 132.0423[M-H+2Na]+177.0858[M-H]-131.035012.60
guanosineC10H13N5O5283.0917[M+H]+284.0989[M-H]-282.08448.57
homoserineC4H9NO3119.0582[M+H]+120.0655[M-H]-118.051013.65
hypoxanthineC5H4N4O136.0385[M+H]+137.0458[M-H]-135.03123.10
inosineC10H12N4O5268.0808[M+H]+269.0880[M-H]-267.07355.43
isoleucineC6H13NO2131.0946[M+H]+132.1019[M-H]-130.08749.71
jasmonic acidC12H18O3210.1256[M+H]+211.1329[M-H]-209.11831.04
lactic acidC3H6O390.0317[M+H]+91.0390[M-H]-89.02445.06
leucineC6H13NO2131.0946[M+H]+132.1019[M-H]-130.08749.32
lysineC6H14N2O2146.1055[M+H]+147.1128[M-H]-145.098317.01
maltoseC12H22O11342.1162[M+Na]+365.1054[M-H]-341.108914.07
mannitolC6H14O6182.0790[M+H]+183.0863[M-H]-181.07189.53
mannosamineC6H13NO5179.0794[M+H]+180.0866[M-H]-178.072114.52
methionineC5H11NO2S149.0510[M+H]+150.0583[M-H]-148.043810.44
MRFA (Met-Arg-Phe-Ala)C23H37N7O5S523.2577[M+H]+524.2650[M-H]-522.250413.61
N-acetyl-aspartic acidC6H9NO5175.0481[M+H]+176.0553[M-H]-174.040814.82
N-acetyl-glutamic acidC7H11NO5189.0637[M+H]+190.0710[M-H]-188.056415.16
N-acetyl-mannosamineC8H15NO6221.0899[M+Na]+244.0792[M-H]-220.08277.15
N-alpha-acetyl-lysineC8H16N2O3188.1161[M+H]+189.1234[M-H]-187.108815.13
n-butylamineC4H11N73.0891[M+H]+74.0964[M-H]-72.08194.17
nicotinamideC6H6N2O122.0480[M+H]+123.0553[M-H]-121.04071.22
nicotinic acidC6H5NO2123.0320[M+H]+124.0393[M-H]-122.02485.63
ornithineC5H12N2O2132.0899[M+H]+133.0972[M-H]-131.082617.04
phenylacetic acidC8H8O2136.0524[M+H]+137.0597[M-H]-135.04525.88
phenylalanineC9H11NO2165.0790[M+H]+166.0863[M-H]-164.07178.98
pipecolic acidC6H11NO2129.0790[M+H]+130.0863[M-H]-128.071710.97
prolineC5H9NO2115.0633[M+H]+116.0706[M-H]-114.056110.92
pyridoxineC8H11NO3169.0739[M+H]+170.0812[M-H]-168.06662.16
raffinoseC18H32O16504.1690[M+H]+505.1763[M-H]-503.161815.53
rhamnoseC6H12O5164.0685[M+H]+165.0757[M-H]-163.06122.80
riboseC5H10O5150.0528[M+H]+151.0601[M-H]-149.04552.75
salicylic acidC7H6O3138.0317[M+H]+139.0390[M-H]-137.02442.20
serineC3H7NO3105.0426[M+H]+106.0499[M-H]-104.035314.31
shikimic acidC7H10O5174.0528[M+H]+175.0601[M-H]-173.045513.41
succinic acidC4H6O4118.0266[M+H]+119.0339[M-H]-117.01939.92
sucroseC12H22O11342.1162[M+Na]+365.1054[M-H]-341.108913.45
syringic acidC9H10O5198.0528[M+H]+199.0601[M-H]-197.04551.59
taurineC2H7NO3S125.0147[M+H]+126.0219[M-H]-124.007412.16
threonineC4H9NO3119.0582[M+H]+120.0655[M-H]-118.051013.49
thymidineC10H14N2O5242.0903[M+H]+243.0975[M-H]-241.08301.60
thymineC5H6N2O2126.0429[M+H]+127.0502[M-H]-125.03571.26
trans-4-hydroxy-prolineC5H9NO3131.0582[M+H]+132.0655[M-H]-130.051013.25
trans-cinnamic acidC9H8O2148.0524[M+H]+149.0597[M-H]-147.04521.15
trehaloseC12H22O11342.1162[M+Na]+365.1054[M-H]-341.108914.21
tryptophanC11H12N2O2204.0899[M+H]+205.0972[M-H]-203.082610.16
uracilC4H4N2O2112.0273[M+H]+113.0346[M-H]-111.02001.39
uridineC9H12N2O6244.0695[M+H]+245.0768[M-H]-243.06232.89
urocanic acidC6H6N2O2138.0429[M+H]+139.0502[M-H]-137.03579.35
valineC5H11NO2117.0790[M+H]+118.0863[M-H]-116.071711.12
vanillic acidC8H8O4168.0423[M+H]+169.0495[M-H]-167.03501.54
vanillinC8H8O3152.0473[M+H]+153.0546[M-H]-151.04010.86
xanthineC5H4N4O2152.0334[M+H]+153.0407[M-H]-151.02612.73
xanthosineC10H12N4O6284.0757[M+H]+285.0830[M-H]-283.06849.78
Table 10. Representative QC Mix for polar HILIC-Z. For each compound, observed adduct in positive and negative mode are listed as well as the observed retention time using this LC-MS/MS method.
  • Note: Some compounds are only detected in a single polarity. Most ionize well at a concentration of 25 µM.

Blank
100% MeOH only (or other solvent matching the resuspension solvent of experimental samples).

Usage: Blank injections are interspersed between each sample injection to monitor background and minimize carryover (e.g. compounds "caught" in the system from the previous injection and detected in the next injection) between samples.
Internal Standard (ISTD) mix
A custom mixture of isotopically labeled (and/or non-biological/synthetic) compounds. These are added (at a specific concentration) to each sample prior to running LC-MS (typically during resuspension).

Usage: (1) Similar to QC mix, injections of ISTD mix only are interspersed throughout the LC-MS run to monitor instrument performance (calibration, intensity, retention time, etc.) as well as adjust compound retention times between runs. (2) Every experimental sample is also resuspended in solvent containing ISTDs. Since these are present in every sample at the same concentration, these can be used to assess individual sample injection properties, including changes in retention time (e.g. due to sample pH, clogging), failed injections, or intensity variations (e.g. due to matrix effects, source fouling, or other factors). Additionally, since concentrations are known, an approximation of concentration for the same compound found in an experimental sample (not isotopically labeled) can be estimated based on ratio.

ABCDEFGHIJK
CompoundFormulaConcentrationMonoisotopic massAdduct (POS)m/z (POS)Adduct (NEG)m/z (NEG)Expected RTPart numberNotes
alanine (U - 13C, 15N)[13C]3H7[15N]O232.5 µM93.0548[M+H]+94.0620[M-H]-92.047513.41767964, Sigma
arginine (U - 13C, 15N)[13C]6H14[15N]4O211 µM184.1199[M+H]+185.1272[M-H]-183.112716.94767964, Sigma
asparagine (U - 13C, 15N)[13C]4H8[15N]2O39.5 µM138.0610[M+H]+139.0683[M-H]-137.053714.37767964, Sigma
aspartic acid (U - 13C, 15N)[13C]4H7[15N]O426 µM138.0480[M+H]+139.0552[M-H]-137.040716.13767964, Sigma
cysteine (U - 13C, 15N)[13C]3H7[15N]O2S10 µM125.0268[M+H]+126.0341[M-H]-124.0196Not detected767964, SigmaNot detected, oxidizes to cystine
cystine (U - 13C, 15N)[13C]6H12[15N]2O4S210 µM248.0380[M+H]+249.0453[M-H]-247.030816.9N/ANot added but a byproduct of cysteine (in amino acid mix)
glutamic acid (U - 13C, 15N)[13C]5H9[15N]O421 µM153.0670[M+H]+154.0742[M-H]-152.059715.94767964, Sigma
glutamine (U - 13C, 15N)[13C]5H10[15N]2O310 µM153.0800[M+H]+154.0873[M-H]-152.072714.31767964, Sigma
glycine (U - 13C, 15N)[13C]2H5[15N]O224 µM78.0358[M+H]+79.0430[M-H]-77.028514.1767964, Sigma
histidine (U - 13C, 15N)[13C]6H9[15N]3O22.5 µM164.0807[M+H]+165.0880[M-H]-163.073414.88767964, Sigma
isoleucine (U - 13C, 15N)[13C]6H13[15N]O210.5 µM138.1118[M+H]+139.1191[M-H]-137.10459.71767964, Sigma
leucine (U - 13C, 15N)[13C]6H13[15N]O222.5 µM138.1118[M+H]+139.1191[M-H]-137.10459.32767964, Sigma
lysine (U - 13C, 15N)[13C]6H14[15N]2O29.5 µM154.1197[M+H]+155.1270[M-H]-153.112417.01767964, Sigma
methionine (U - 13C, 15N)[13C]5H11[15N]O2S4.5 µM155.0649[M+H]+156.0721[M-H]-154.057610.44767964, Sigma
phenylalanine (U - 13C, 15N)[13C]9H11[15N]O28.5 µM175.1062[M+H]+176.1135[M-H]-174.09898.98767964, Sigma
proline (U - 13C, 15N)[13C]5H9[15N]O29.5 µM121.0771[M+H]+122.0844[M-H]-120.069910.92767964, Sigma
serine (U - 13C, 15N)[13C]3H7[15N]O314 µM109.0497[M+H]+110.0570[M-H]-108.042414.31767964, Sigma
threonine (U - 13C, 15N)[13C]4H9[15N]O314 µM124.0687[M+H]+125.0760[M-H]-123.061413.49767964, Sigma
tryptophan (U - 13C, 15N)[13C]11H12[15N]2O210 µM217.1209[M+H]+218.1281[M-H]-216.113610.16767964, Sigma
tyrosine (U - 13C, 15N)[13C]9H11[15N]O36.5 µM191.1011[M+H]+192.1084[M-H]-190.093811.86767964, Sigma
valine (U - 13C, 15N)[13C]5H11[15N]O214.5 µM123.0928[M+H]+124.1001[M-H]-122.085511.12767964, Sigma
mannitol (U - 13C)[13C]6H14O610 µg/mL188.0992[M+H]+189.1064[M-H]-187.09199.53ALD-030, Omicron Biochemicals
trehalose (U - 13C)[13C]12H22O1110 ug/mL354.1565[M+Na]+377.1457[M-H]-353.149214.44TRE-002, Omicron Biochemicals
adenine (U - 15N)C5H5[15N]54 µg/mL140.0397[M+H]+141.0469[M-H]-139.03242.56NLM-6924, Cambridge Isotope Labs
hypoxanthine (U - 15N)C5H4[15N]4O3 µg/mL140.0267[M+H]+141.0339[M-H]-139.01943.1NLM-8500, Cambridge Isotope Labs
uracil (U - 13C, 15N)[13C]4H4O2[15N]22 µg/mL118.0348[M+H]+119.0420[M-H]-117.02751.39CNLM-3917, Cambridge Isotope Labs
inosine (U - 15N)C10H12[15N]4O55.5 µg/mL272.0689[M+H]+273.0762[M-H]-271.06165.43NLM-4264, Cambridge Isotope Labs
cytosine (13C2, 15N3)C2[13C]2H5[15N]3O5 µg/mL116.0411[M+H]+117.0483[M-H]-115.03384.83492108, Sigma
guanine (U - 15N)C5H5[15N]5O2 µg/mL156.0346[M+H]+157.0419[M-H]-155.02736.27NLM-6926, Cambridge Isotope Labs
thymine (U - 13C, 15N)[13C]5H6[15N]2O22.5 µg/mL133.0538[M+H]+134.0610[M-H]-132.04651.26CNLM-6945, Cambridge Isotope Labs
2-amino-3-bromo-5-methylbenzoic acid (ABMBA) (Br-nat)C8H8BrNO21 µg/mL228.9738[M+H]+229.9811[M-H]-227.96661.2631531, Sigma
Table 11. Representative ISTD Mix used for resuspension. For each compound, observed adduct in positive and negative ion mode are listed as well as the observed retention time using this LC-MS/MS method. Concentrations listed are the typical concentrations used in this protocol.
  • For the Sigma amino acid mix (#767964, Sigma), the final concentration varies for each compound between lots, but is usually very similar between lots. Average concentration is ~15 µM for all compounds across the Sigma amino acid mix.
  • For stable isotope labeling studies with 13C, typically only the 13C-15N amino acid mixture and ABMBA (synthetic and contains bromine - isobaric) are used as internal standards. Similar adjustments are made for other types of stable isotope labeling experiments based on type of labeling and overall experimental design.

Experimental Sample Vial
Samples typically consist of metabolite extracts (or a compound standard at a specified concentration) resuspended in solvent, usually 100% MeOH, and containing a mixture of isotopically labeled internal standards (see example ISTD mix above).

Usage: To profile metabolites in a sample and/or annotate the retention time, ionization characteristics (m/z for an adduct) and fragmentation spectra of a compound.

LC System Preparation
To prepare the LC, column compartment (40 ºC) and autosampler temperatures (4 ºC) are set and monitored until stable. The LC binary pump is typically prepared by purging 100% mobile phase A, 50/0 mobile phase A/B, then 100% B, each for 7 minutes at a flow rate of 5 mL/min. A flow rate of 0.45 mL/min of 100% B is maintained while the UHPLC system is checked for leaks or clogs. Backpressure is monitored until stable at ~130 bar. The HILIC column is equilibrated by performing 3-10 injections using the method gradients provided in Table 6.

Mass Spectrometer Preparation
Prior to data acquisition, the mass spectrometer is calibrated using standard calibration procedures available in the Thermo XCalibur operating software. ESI needle position is optimized relative to the source to achieve stable and acceptable ion intensity levels.

Calibration procedure for QE and QE-HF. Here, calibration is performed in positive mode with Pierce™ LTQ Velos ESI Positive Ion Calibration Solution (#88323, Thermo Scientific) and negative mode with Pierce™ Negative Ion Calibration Solution (#88324, Thermo Scientific) using direct injection from a syringe pump. Standard calibration is then followed by a custom low mass calibration procedure to ensure compounds near 100 m/z are also well-calibrated.

Calibration procedure for Exp120 and IQX. Here, calibration is performed in both positive and negative ionization mode with Pierce™ FlexMix Calibration Solution (#A39239, Thermo Scientific) using direct injection from a syringe pump and insertion of the low-flow ESI needle into the source housing. Custom low mass calibration procedures are not necessary using these systems. The system can be re-calibrated periodically during data acquisition using an automated point calibration (EasyIC) with the internal calibrant compound fluoroanthene. Prior to starting an LC-MS/MS run for data collection, the low-flow needle is replaced by the high-flow needle.

Note: For the IQX, an auto-calibration option can be performed using the Auto-ready Ion Source. This uses a less concentrated calibration mix (Pierce™ FlexMix Calibration Solution for Auto-Ready Mass Spectrometers, #A51739, Thermo Scientific) and eliminates the need to switch between low- and high-flow ESI needles.

LC-MS/MS Data Collection
In a typical LC-MS/MS run, an injection volume of 2-3 µL for each sample is used. Each sample is run in positive and negative ionization mode, with an injection blank of 100% methanol interspersed between each sample, replaced by an ISTD mix interspersed every 3 samples and a QC mix every 9-15 samples. Sample injection order is randomized between groups of replicate 1, then replicate 2, etc. Prior to starting a full experimental run, at least 4 injection blanks and several QC and ISTD injections are performed to ensure column and system equilibration and to verify that data is being acquired as expected. ISTD mix compounds are regularly monitored throughout LC-MS runs to assess drops in intensity, retention time shifts or increases in m/z ppm error, and performing the appropriate cleaning, re-calibration, maintenance or other troubleshooting as needed.

Protocol references
1 - https://massive.ucsd.edu/

2 - Eric W. Deutsch, Attila Csordas, Zhi Sun, Andrew Jarnuczak, Yasset Perez-Riverol, Tobias Ternent, David S. Campbell, Manuel Bernal-Llinares, Shujiro Okuda, Shin Kawano, Robert L. Moritz, Jeremy J. Carver, Mingxun Wang, Yasushi Ishihama, Nuno Bandeira, Henning Hermjakob, Juan Antonio Vizcaíno, The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition, Nucleic Acids Research, Volume 45, Issue D1, January 2017, Pages D1100–D1106, https://doi.org/10.1093/nar/gkw936
3 - Wang, M., Carver, J., Phelan, V. et al. Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nat Biotechnol 34, 828–837 (2016). https://doi.org/10.1038/nbt.3597