TITLE: JGI/LBNL Metabolomics - Standard LC-MS/MS ESI Method - Nonpolar C18
AUTHORS: Katherine B. Louie, Suzanne Kosina, Thomas Harwood, Meghana Faltane, Marie Lynde, Benjamin P. Bowen, Trent Northen

[DESCRIPTION]
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 non-polar metabolites (e.g. secondary metabolites, etc) from sample extracts using reverse phase chromatography (C18) 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 2014, 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.

[STEPS]
SECTION: Mass Spectrometer Preparation
13. 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.

SECTION: LC-MS/MS Method Parameters
6. LIQUID CHROMATOGRAPHY

 

Column
Agilent ZORBAX Eclipse Plus C18, Rapid Resolution HD, 2.1 x 50 mm, 1.8 μm, 95 Å (Agilent, 959757-902); max pressure 1200 bar

Column Temperature 60 °C      
Autosampler Temperature  4 °C 

 

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  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.
SECTION: Sample Vial Preparation
8. 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.  Pre-made mixes can also be used (e.g. AOAC QuEChERS QC Spike Mix, #31999, Restek).

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.

 

SECTION: Instrumentation / Equipment
2. Mass Spectrometry Instrumentation

 
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.
 
SECTION: Overview
1. 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 analyze non-polar metabolites (e.g. secondary metabolites, etc) from sample extracts using reverse phase chromatography (C18) coupled to a Thermo Orbitrap Mass Spectrometer with ESI source. 

SECTION: LC System Preparation
12. To prepare the LC, column compartment (60 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 0.40 mL/min of 100% B is maintained while the UHPLC system is checked for leaks or clogs.  Backpressure is monitored until stable.  The HILIC column is equilibrated by performing 3-10 injections using the method gradients provided in Table 6.

7. MASS SPECTROMETRY

Source settings
 

MS1 Settings
 

Collection time: 12 minutes

MS2 Settings
 

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.

SECTION: Instrumentation / Equipment
3. UHPLC System

 

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.
SECTION: Sample Vial Preparation
10. 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.

 

SECTION: Sample Vial Preparation
9. 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.
SECTION: Sample Vial Preparation
11. 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.

SECTION: Instrumentation / Equipment
4. UHPLC Column information


 


SECTION: Instrumentation / Equipment
5. Chemicals / solvents
 


SECTION: LC-MS/MS Data Collection
14. 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.