Oct 01, 2024
JGI/LBNL Metabolomics - Standard LC-MS/MS ESI Method - Polar HILIC-Z
- Katherine B. Louie1,
- Suzanne Kosina2,
- Thomas Harwood1,
- Meghana Faltane1,
- Marie Lynde1,
- Benjamin P. 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
Protocol Citation: Katherine B. Louie, Suzanne Kosina, Thomas Harwood, Meghana Faltane, Marie Lynde, Benjamin P. 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
Funders Acknowledgement:
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.
Overview
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
Instrumentation / Equipment
Mass Spectrometry Instrumentation
| A | B | C | D | |
| Mass Spectrometer | Source / Probe | ESI needle (calibration) | ESI needle (running samples) | |
| Thermo Q Exactive HF Orbitrap (QE-HF) | Thermo Ion Max API Source w/ H-ESI II probe | 32G 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 probe | 32G 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 probe | 35G 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 probe | 35G 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
| A | B | C | D | |
| Module Name | Module Type | Part Numbers | Alternative Part Numbers | |
| 1290 Infinity DAD* | Diode Array Detector | G4212A | G7115A, G7117BR | |
| 1200 Infinity Series TCC | Column Compartment | G1316A | G7116B | |
| 1290 Infinity Sampler | Autosampler | G4226A | G7167B - Multisampler w/ thermostat | |
| 1290 Infinity Thermostat | Autosampler Thermostat | G1330B | G7167B - Multisampler w/ thermostat | |
| 1290 Infinity Bin Pump | Binary Pump | G4220A | G7120A |
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
| A | B | |
| Column name | InfinityLab Poroshell 120 HILIC-Z | |
| Part # | #683775-924 | |
| Manufacturer | Agilent | |
| Column chemistry | HILIC-Z, zwitterionic | |
| Inner Diameter (ID) | 2.1 mm | |
| Length | 150 mm | |
| Particle size | 2.7 µm | |
| Pore size | 100 Å | |
| Max pressure | 600 bar | |
| pH range | 2-12 | |
| Max temperature | 80 ºC @ pH 7; 35 ºC @ pH 2-12 |
Table 3. UHPLC column information.
Chemicals / solvents
| A | B | |
| Chemicals / solvents | Product 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
LC-MS/MS Method Parameters
LIQUID CHROMATOGRAPHY
| A | B | |
| Mobile Phase A | 99.8% H2O and 0.2% acetic acid, w/ 5 mM ammonium acetate and 5 µM methylene-di-phosphonic acid | |
| Mobile Phase B | 99.8% 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 40 °C
Autosampler Temperature 4 °C
| A | B | C | D | E | |
| Time (min) | Flow (mL/min) | %A | %B | minutes for segment | |
| 0 | 0.45 | 0 | 100 | ||
| 1 | 0.45 | 0 | 100 | 1 | |
| 11 | 0.45 | 11 | 89 | 10 | |
| 15.75 | 0.45 | 30 | 70 | 4.75 | |
| 16.25 | 0.45 | 80 | 20 | 0.5 | |
| 18.5 | 0.45 | 80 | 20 | 2.25 | |
| 18.6 | 0.45 | 0 | 100 | 0.1 | |
| 21 | 0.45 | 0 | 100 | 2.4 |
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
| A | B | C | D | E | |
| Parameter | QE-HF | QE | Exp120 | IQX | |
| Sheath Gas Flow Rate (au) | 55 | 55 | 50 | 50 | |
| Auxillary Gas Flow Rate (au) | 20 | 20 | 10 | 10 | |
| Sweep Gas Flow Rate (au) | 2 | 2 | 1 | 1 | |
| Spray Voltage (V) - POS | 3000 | 3000 | 3500 | 3500 | |
| Spray Voltage (V) - NEG | 3000 | 3000 | 2500 | 2500 | |
| Capillary Temperature (°C) | 400 | 400 | 325 | 325 | |
| Vaporizer Temperature (°C) | N/A | N/A | 300 | 300 | |
| S-Lens RF Level (%) | 50 | 50 | 70 | 50 |
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
| A | B | C | D | E | |
| Parameter | QE-HF | QE | Exp120 | IQX | |
| Microscans | 1 | 1 | 1 | 1 | |
| Resolution | 60,000 | 70,000 | 60,000 | 60,000 | |
| AGC Target | 3e6 | 3e6 | Standard (100%) | 1e5 | |
| Maximum IT (ms) | 100 | 100 | Auto | 118 | |
| Scan range (m/z) | 70 - 1050 | 70 - 1050 | 70 - 1050 | 70 - 1050 | |
| Spectrum data type | Centroid | Centroid | Centroid | Centroid |
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
| A | B | C | D | E | |
| Parameter | QE-HF | QE | Exp120 | IQX | |
| Microscans | 1 | 1 | 1 | 1 | |
| Resolution | 15,000 | 17,500 | 15,000 | 15,000 | |
| AGC Target | 1e5 | 1e5 | Standard (100%) | 5e4 | |
| Maximum IT (ms) | 50 | 50 | Auto | 22 | |
| Loop count | 2 (or 4) | 2 (or 4) | 4 | N/A | |
| Cycle time | N/A | N/A | N/A | 0.8 sec | |
| MSX count | 1 | 1 | 1 | 1 | |
| TopN | 2 (or 4) | 2 (or 4) | 4 | 10-15 | |
| Exclusion duration (sec) | 7 - 10 | 7 - 10 | 4 | 5 | |
| 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
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.
| A | B | C | D | E | F | G | H | |
| Compound | Formula | Monoisotopic mass | Adduct (POS) | m/z (POS) | Adduct (NEG) | m/z (NEG) | Expected RT | |
| 1-methyladenosine | C11H15N5O4 | 281.1124 | [M+H]+ | 282.1197 | [M-H]- | 280.1051 | 10.78 | |
| 2,4-dihydroxypteridine | C6H4N4O2 | 164.0334 | [M+H]+ | 165.0407 | [M-H]- | 163.0261 | 1.27 | |
| 2'-deoxyadenosine | C10H13N5O3 | 251.1018 | [M+H]+ | 252.1091 | [M-H]- | 250.0946 | 2.23 | |
| 2'-deoxyguanosine | C10H13N5O4 | 267.0968 | [M+H]+ | 268.1040 | [M-H]- | 266.0895 | 6.87 | |
| 2-hydroxyphenylacetic acid | C8H8O3 | 152.0473 | [M+H]+ | 153.0546 | [M-H]- | 151.0401 | 1.62 | |
| 2-oxovaleric acid | C5H8O3 | 116.0473 | [M+H]+ | 117.0546 | [M-H]- | 115.0401 | 4.45 | |
| 4-coumaric acid | C9H8O3 | 164.0473 | [M+H]+ | 165.0546 | [M-H]- | 163.0401 | 1.51 | |
| 4-guanidinobutanoic acid | C5H11N3O2 | 145.0851 | [M+H]+ | 146.0924 | [M-H]- | 144.0779 | 13.86 | |
| 4-methoxyphenylacetic acid | C9H10O3 | 166.0630 | [M+H]+ | 167.0703 | [M-H]- | 165.0557 | 1.07 | |
| 5-methylcytosine | C5H7N3O | 125.0589 | [M+H]+ | 126.0662 | [M-H]- | 124.0516 | 4.42 | |
| 5-oxo-proline | C5H7NO3 | 129.0426 | [M+H]+ | 130.0499 | [M-H]- | 128.0353 | 11.65 | |
| ABMBA (2-amino-3-bromo-5-methylbenzoic acid) | C8H8BrNO2 | 228.9738 | [M+H]+ | 229.9811 | [M-H]- | 227.9666 | 1.20 | |
| abscisic acid | C15H20O4 | 264.1362 | [M+H]+ | 265.1434 | [M-H]- | 263.1289 | 1.15 | |
| acetylcholine | C7NH16O2+ | 146.1176 | [M]+ | 146.1176 | [M-2H]- | 144.1030 | 1.96 | |
| adenine | C5H5N5 | 135.0545 | [M+H]+ | 136.0618 | [M-H]- | 134.0472 | 2.56 | |
| adenosine | C10H13N5O4 | 267.0968 | [M+H]+ | 268.1040 | [M-H]- | 266.0895 | 3.09 | |
| alanine | C3H7NO2 | 89.0477 | [M+H]+ | 90.0550 | [M-H]- | 88.0404 | 13.41 | |
| alpha-glucose | C6H12O6 | 180.0634 | [M+H]+ | 181.0707 | [M-2H]2- | 89.0707 | 5.06 | |
| alpha-ketoglutaric acid | C5H6O5 | 146.0215 | [M+H]+ | 147.0288 | [M-H]- | 145.0142 | 14.51 | |
| arginine | C6H14N4O2 | 174.1117 | [M+H]+ | 175.1190 | [M-H]- | 173.1044 | 16.94 | |
| asparagine | C4H8N2O3 | 132.0535 | [M+H]+ | 133.0608 | [M-H]- | 131.0462 | 14.37 | |
| aspartic acid | C4H7NO4 | 133.0375 | [M+H]+ | 134.0448 | [M-H]- | 132.0302 | 16.13 | |
| benzoic acid | C7H6O2 | 122.0368 | [M+H]+ | 123.0441 | [M-H]- | 121.0295 | 1.27 | |
| betaine | C5H12NO2+ | 118.0863 | [M]+ | 118.0863 | [M-2H]- | 116.0717 | 7.91 | |
| caffeic acid | C9H8O4 | 180.0423 | [M+H]+ | 181.0495 | [M-H]- | 179.0350 | 3.22 | |
| caffeine | C8H10N4O2 | 194.0804 | [M+H]+ | 195.0877 | [M-H]- | 193.0731 | 0.88 | |
| carnitine | C7H16NO3+ | 162.1125 | [M+H]+ | 162.1125 | [M-H]- | 160.0979 | 13.29 | |
| cis-4-hydroxy-proline | C5H9NO3 | 131.0582 | [M+H]+ | 132.0655 | [M-H]- | 130.0510 | 13.67 | |
| citrulline | C6H13N3O3 | 175.0957 | [M+H]+ | 176.1030 | [M-H]- | 174.0884 | 15.09 | |
| creatine | C4H9N3O2 | 131.0695 | [M+H]+ | 132.0768 | [M-H]- | 130.0622 | 13.39 | |
| cysteic acid | C3H7NO5S | 169.0045 | [M+H]+ | 170.0118 | [M-H]- | 167.9972 | 14.54 | |
| cytidine | C9H13N3O5 | 243.0855 | [M+H]+ | 244.0928 | [M-H]- | 242.0782 | 6.93 | |
| cytosine | C4H5N3O | 111.0433 | [M+H]+ | 112.0505 | [M-H]- | 110.0360 | 4.83 | |
| deoxycytidine | C9H13N3O4 | 227.0906 | [M+H]+ | 228.0979 | [M-H]- | 226.0833 | 5.59 | |
| deoxyuridine | C9H12N2O5 | 228.0746 | [M+H]+ | 229.0819 | [M-H]- | 227.0673 | 1.88 | |
| ectoine | C6H10N2O2 | 142.0742 | [M+H]+ | 143.0815 | [M-H]- | 141.0670 | 12.50 | |
| fumaric acid | C4H4O4 | 116.0110 | [M+H]+ | 117.0182 | [M-H]- | 115.0037 | 16.31 | |
| gamma-aminobutyric acid | C4H9NO2 | 103.0633 | [M+H]+ | 104.0706 | [M-H]- | 102.0561 | 14.39 | |
| glutamic acid | C5H9NO4 | 147.0532 | [M+H]+ | 148.0604 | [M-H]- | 146.0459 | 15.94 | |
| glutamine | C5H10N2O3 | 146.0691 | [M+H]+ | 147.0764 | [M-H]- | 145.0619 | 14.31 | |
| glutaric acid | C5H8O4 | 132.0423 | [M-H+2Na]+ | 177.0858 | [M-H]- | 131.0350 | 12.60 | |
| guanosine | C10H13N5O5 | 283.0917 | [M+H]+ | 284.0989 | [M-H]- | 282.0844 | 8.57 | |
| homoserine | C4H9NO3 | 119.0582 | [M+H]+ | 120.0655 | [M-H]- | 118.0510 | 13.65 | |
| hypoxanthine | C5H4N4O | 136.0385 | [M+H]+ | 137.0458 | [M-H]- | 135.0312 | 3.10 | |
| inosine | C10H12N4O5 | 268.0808 | [M+H]+ | 269.0880 | [M-H]- | 267.0735 | 5.43 | |
| isoleucine | C6H13NO2 | 131.0946 | [M+H]+ | 132.1019 | [M-H]- | 130.0874 | 9.71 | |
| jasmonic acid | C12H18O3 | 210.1256 | [M+H]+ | 211.1329 | [M-H]- | 209.1183 | 1.04 | |
| lactic acid | C3H6O3 | 90.0317 | [M+H]+ | 91.0390 | [M-H]- | 89.0244 | 5.06 | |
| leucine | C6H13NO2 | 131.0946 | [M+H]+ | 132.1019 | [M-H]- | 130.0874 | 9.32 | |
| lysine | C6H14N2O2 | 146.1055 | [M+H]+ | 147.1128 | [M-H]- | 145.0983 | 17.01 | |
| maltose | C12H22O11 | 342.1162 | [M+Na]+ | 365.1054 | [M-H]- | 341.1089 | 14.07 | |
| mannitol | C6H14O6 | 182.0790 | [M+H]+ | 183.0863 | [M-H]- | 181.0718 | 9.53 | |
| mannosamine | C6H13NO5 | 179.0794 | [M+H]+ | 180.0866 | [M-H]- | 178.0721 | 14.52 | |
| methionine | C5H11NO2S | 149.0510 | [M+H]+ | 150.0583 | [M-H]- | 148.0438 | 10.44 | |
| MRFA (Met-Arg-Phe-Ala) | C23H37N7O5S | 523.2577 | [M+H]+ | 524.2650 | [M-H]- | 522.2504 | 13.61 | |
| N-acetyl-aspartic acid | C6H9NO5 | 175.0481 | [M+H]+ | 176.0553 | [M-H]- | 174.0408 | 14.82 | |
| N-acetyl-glutamic acid | C7H11NO5 | 189.0637 | [M+H]+ | 190.0710 | [M-H]- | 188.0564 | 15.16 | |
| N-acetyl-mannosamine | C8H15NO6 | 221.0899 | [M+Na]+ | 244.0792 | [M-H]- | 220.0827 | 7.15 | |
| N-alpha-acetyl-lysine | C8H16N2O3 | 188.1161 | [M+H]+ | 189.1234 | [M-H]- | 187.1088 | 15.13 | |
| n-butylamine | C4H11N | 73.0891 | [M+H]+ | 74.0964 | [M-H]- | 72.0819 | 4.17 | |
| nicotinamide | C6H6N2O | 122.0480 | [M+H]+ | 123.0553 | [M-H]- | 121.0407 | 1.22 | |
| nicotinic acid | C6H5NO2 | 123.0320 | [M+H]+ | 124.0393 | [M-H]- | 122.0248 | 5.63 | |
| ornithine | C5H12N2O2 | 132.0899 | [M+H]+ | 133.0972 | [M-H]- | 131.0826 | 17.04 | |
| phenylacetic acid | C8H8O2 | 136.0524 | [M+H]+ | 137.0597 | [M-H]- | 135.0452 | 5.88 | |
| phenylalanine | C9H11NO2 | 165.0790 | [M+H]+ | 166.0863 | [M-H]- | 164.0717 | 8.98 | |
| pipecolic acid | C6H11NO2 | 129.0790 | [M+H]+ | 130.0863 | [M-H]- | 128.0717 | 10.97 | |
| proline | C5H9NO2 | 115.0633 | [M+H]+ | 116.0706 | [M-H]- | 114.0561 | 10.92 | |
| pyridoxine | C8H11NO3 | 169.0739 | [M+H]+ | 170.0812 | [M-H]- | 168.0666 | 2.16 | |
| raffinose | C18H32O16 | 504.1690 | [M+H]+ | 505.1763 | [M-H]- | 503.1618 | 15.53 | |
| rhamnose | C6H12O5 | 164.0685 | [M+H]+ | 165.0757 | [M-H]- | 163.0612 | 2.80 | |
| ribose | C5H10O5 | 150.0528 | [M+H]+ | 151.0601 | [M-H]- | 149.0455 | 2.75 | |
| salicylic acid | C7H6O3 | 138.0317 | [M+H]+ | 139.0390 | [M-H]- | 137.0244 | 2.20 | |
| serine | C3H7NO3 | 105.0426 | [M+H]+ | 106.0499 | [M-H]- | 104.0353 | 14.31 | |
| shikimic acid | C7H10O5 | 174.0528 | [M+H]+ | 175.0601 | [M-H]- | 173.0455 | 13.41 | |
| succinic acid | C4H6O4 | 118.0266 | [M+H]+ | 119.0339 | [M-H]- | 117.0193 | 9.92 | |
| sucrose | C12H22O11 | 342.1162 | [M+Na]+ | 365.1054 | [M-H]- | 341.1089 | 13.45 | |
| syringic acid | C9H10O5 | 198.0528 | [M+H]+ | 199.0601 | [M-H]- | 197.0455 | 1.59 | |
| taurine | C2H7NO3S | 125.0147 | [M+H]+ | 126.0219 | [M-H]- | 124.0074 | 12.16 | |
| threonine | C4H9NO3 | 119.0582 | [M+H]+ | 120.0655 | [M-H]- | 118.0510 | 13.49 | |
| thymidine | C10H14N2O5 | 242.0903 | [M+H]+ | 243.0975 | [M-H]- | 241.0830 | 1.60 | |
| thymine | C5H6N2O2 | 126.0429 | [M+H]+ | 127.0502 | [M-H]- | 125.0357 | 1.26 | |
| trans-4-hydroxy-proline | C5H9NO3 | 131.0582 | [M+H]+ | 132.0655 | [M-H]- | 130.0510 | 13.25 | |
| trans-cinnamic acid | C9H8O2 | 148.0524 | [M+H]+ | 149.0597 | [M-H]- | 147.0452 | 1.15 | |
| trehalose | C12H22O11 | 342.1162 | [M+Na]+ | 365.1054 | [M-H]- | 341.1089 | 14.21 | |
| tryptophan | C11H12N2O2 | 204.0899 | [M+H]+ | 205.0972 | [M-H]- | 203.0826 | 10.16 | |
| uracil | C4H4N2O2 | 112.0273 | [M+H]+ | 113.0346 | [M-H]- | 111.0200 | 1.39 | |
| uridine | C9H12N2O6 | 244.0695 | [M+H]+ | 245.0768 | [M-H]- | 243.0623 | 2.89 | |
| urocanic acid | C6H6N2O2 | 138.0429 | [M+H]+ | 139.0502 | [M-H]- | 137.0357 | 9.35 | |
| valine | C5H11NO2 | 117.0790 | [M+H]+ | 118.0863 | [M-H]- | 116.0717 | 11.12 | |
| vanillic acid | C8H8O4 | 168.0423 | [M+H]+ | 169.0495 | [M-H]- | 167.0350 | 1.54 | |
| vanillin | C8H8O3 | 152.0473 | [M+H]+ | 153.0546 | [M-H]- | 151.0401 | 0.86 | |
| xanthine | C5H4N4O2 | 152.0334 | [M+H]+ | 153.0407 | [M-H]- | 151.0261 | 2.73 | |
| xanthosine | C10H12N4O6 | 284.0757 | [M+H]+ | 285.0830 | [M-H]- | 283.0684 | 9.78 |
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.
| A | B | C | D | E | F | G | H | I | J | K | |
| Compound | Formula | Concentration | Monoisotopic mass | Adduct (POS) | m/z (POS) | Adduct (NEG) | m/z (NEG) | Expected RT | Part number | Notes | |
| alanine (U - 13C, 15N) | [13C]3H7[15N]O2 | 32.5 µM | 93.0548 | [M+H]+ | 94.0620 | [M-H]- | 92.0475 | 13.41 | 767964, Sigma | ||
| arginine (U - 13C, 15N) | [13C]6H14[15N]4O2 | 11 µM | 184.1199 | [M+H]+ | 185.1272 | [M-H]- | 183.1127 | 16.94 | 767964, Sigma | ||
| asparagine (U - 13C, 15N) | [13C]4H8[15N]2O3 | 9.5 µM | 138.0610 | [M+H]+ | 139.0683 | [M-H]- | 137.0537 | 14.37 | 767964, Sigma | ||
| aspartic acid (U - 13C, 15N) | [13C]4H7[15N]O4 | 26 µM | 138.0480 | [M+H]+ | 139.0552 | [M-H]- | 137.0407 | 16.13 | 767964, Sigma | ||
| cysteine (U - 13C, 15N) | [13C]3H7[15N]O2S | 10 µM | 125.0268 | [M+H]+ | 126.0341 | [M-H]- | 124.0196 | Not detected | 767964, Sigma | Not detected, oxidizes to cystine | |
| cystine (U - 13C, 15N) | [13C]6H12[15N]2O4S2 | 10 µM | 248.0380 | [M+H]+ | 249.0453 | [M-H]- | 247.0308 | 16.9 | N/A | Not added but a byproduct of cysteine (in amino acid mix) | |
| glutamic acid (U - 13C, 15N) | [13C]5H9[15N]O4 | 21 µM | 153.0670 | [M+H]+ | 154.0742 | [M-H]- | 152.0597 | 15.94 | 767964, Sigma | ||
| glutamine (U - 13C, 15N) | [13C]5H10[15N]2O3 | 10 µM | 153.0800 | [M+H]+ | 154.0873 | [M-H]- | 152.0727 | 14.31 | 767964, Sigma | ||
| glycine (U - 13C, 15N) | [13C]2H5[15N]O2 | 24 µM | 78.0358 | [M+H]+ | 79.0430 | [M-H]- | 77.0285 | 14.1 | 767964, Sigma | ||
| histidine (U - 13C, 15N) | [13C]6H9[15N]3O2 | 2.5 µM | 164.0807 | [M+H]+ | 165.0880 | [M-H]- | 163.0734 | 14.88 | 767964, Sigma | ||
| isoleucine (U - 13C, 15N) | [13C]6H13[15N]O2 | 10.5 µM | 138.1118 | [M+H]+ | 139.1191 | [M-H]- | 137.1045 | 9.71 | 767964, Sigma | ||
| leucine (U - 13C, 15N) | [13C]6H13[15N]O2 | 22.5 µM | 138.1118 | [M+H]+ | 139.1191 | [M-H]- | 137.1045 | 9.32 | 767964, Sigma | ||
| lysine (U - 13C, 15N) | [13C]6H14[15N]2O2 | 9.5 µM | 154.1197 | [M+H]+ | 155.1270 | [M-H]- | 153.1124 | 17.01 | 767964, Sigma | ||
| methionine (U - 13C, 15N) | [13C]5H11[15N]O2S | 4.5 µM | 155.0649 | [M+H]+ | 156.0721 | [M-H]- | 154.0576 | 10.44 | 767964, Sigma | ||
| phenylalanine (U - 13C, 15N) | [13C]9H11[15N]O2 | 8.5 µM | 175.1062 | [M+H]+ | 176.1135 | [M-H]- | 174.0989 | 8.98 | 767964, Sigma | ||
| proline (U - 13C, 15N) | [13C]5H9[15N]O2 | 9.5 µM | 121.0771 | [M+H]+ | 122.0844 | [M-H]- | 120.0699 | 10.92 | 767964, Sigma | ||
| serine (U - 13C, 15N) | [13C]3H7[15N]O3 | 14 µM | 109.0497 | [M+H]+ | 110.0570 | [M-H]- | 108.0424 | 14.31 | 767964, Sigma | ||
| threonine (U - 13C, 15N) | [13C]4H9[15N]O3 | 14 µM | 124.0687 | [M+H]+ | 125.0760 | [M-H]- | 123.0614 | 13.49 | 767964, Sigma | ||
| tryptophan (U - 13C, 15N) | [13C]11H12[15N]2O2 | 10 µM | 217.1209 | [M+H]+ | 218.1281 | [M-H]- | 216.1136 | 10.16 | 767964, Sigma | ||
| tyrosine (U - 13C, 15N) | [13C]9H11[15N]O3 | 6.5 µM | 191.1011 | [M+H]+ | 192.1084 | [M-H]- | 190.0938 | 11.86 | 767964, Sigma | ||
| valine (U - 13C, 15N) | [13C]5H11[15N]O2 | 14.5 µM | 123.0928 | [M+H]+ | 124.1001 | [M-H]- | 122.0855 | 11.12 | 767964, Sigma | ||
| mannitol (U - 13C) | [13C]6H14O6 | 10 µg/mL | 188.0992 | [M+H]+ | 189.1064 | [M-H]- | 187.0919 | 9.53 | ALD-030, Omicron Biochemicals | ||
| trehalose (U - 13C) | [13C]12H22O11 | 10 ug/mL | 354.1565 | [M+Na]+ | 377.1457 | [M-H]- | 353.1492 | 14.44 | TRE-002, Omicron Biochemicals | ||
| adenine (U - 15N) | C5H5[15N]5 | 4 µg/mL | 140.0397 | [M+H]+ | 141.0469 | [M-H]- | 139.0324 | 2.56 | NLM-6924, Cambridge Isotope Labs | ||
| hypoxanthine (U - 15N) | C5H4[15N]4O | 3 µg/mL | 140.0267 | [M+H]+ | 141.0339 | [M-H]- | 139.0194 | 3.1 | NLM-8500, Cambridge Isotope Labs | ||
| uracil (U - 13C, 15N) | [13C]4H4O2[15N]2 | 2 µg/mL | 118.0348 | [M+H]+ | 119.0420 | [M-H]- | 117.0275 | 1.39 | CNLM-3917, Cambridge Isotope Labs | ||
| inosine (U - 15N) | C10H12[15N]4O5 | 5.5 µg/mL | 272.0689 | [M+H]+ | 273.0762 | [M-H]- | 271.0616 | 5.43 | NLM-4264, Cambridge Isotope Labs | ||
| cytosine (13C2, 15N3) | C2[13C]2H5[15N]3O | 5 µg/mL | 116.0411 | [M+H]+ | 117.0483 | [M-H]- | 115.0338 | 4.83 | 492108, Sigma | ||
| guanine (U - 15N) | C5H5[15N]5O | 2 µg/mL | 156.0346 | [M+H]+ | 157.0419 | [M-H]- | 155.0273 | 6.27 | NLM-6926, Cambridge Isotope Labs | ||
| thymine (U - 13C, 15N) | [13C]5H6[15N]2O2 | 2.5 µg/mL | 133.0538 | [M+H]+ | 134.0610 | [M-H]- | 132.0465 | 1.26 | CNLM-6945, Cambridge Isotope Labs | ||
| 2-amino-3-bromo-5-methylbenzoic acid (ABMBA) (Br-nat) | C8H8BrNO2 | 1 µg/mL | 228.9738 | [M+H]+ | 229.9811 | [M-H]- | 227.9666 | 1.2 | 631531, 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
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
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
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
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