LC-MS metabolomics guide-CIBC collaborations

Preface

Powers’ Laboratory Guide for Metabolomics Experiments (written by Aline De Lima Liete, Amith Maroli editted by Isin Tuna Sakallioglu)

This guide provides a good practical foundation to guide our users through basics steps involved in metabolomic analysis by liquid chromatography mass spectrometry, gas chromatography mass spectrometry. These guidelines can be applied to different types of samples to improve the quality of the results.

Please reach out Isin Tuna Sakallioglu from isin-tuna.sakallioglu@huskers.unl.edu to see the complete PDF file with figures. Further questions while following the protocols could be asked to Dr. Liete and Dr. Maroli. This site has similar information.

Important Notes

Collaborator Expense

MS analysis are very expensive labor we should let the colloborators know about the details of the cost before starting the project! You may see chapter I for the details. NOTE: IT IS ESSENTAIL TO SEND AN EXCEL SPREADSHEET THAT HAS DETAILS OF ALL SAMPLES AND DETAILED TOTAL COST OF THE PROJECT TO THE COLLOBORATOR. This is to guarantee we have organized appropriate storage facility well in advance of receiving samples and to avoid any kind of financial conflict with the colloborator.

Avoid

You MUST completely AVOID! STABILIZERS: DO NOT USE any glycerol, PEG or related stabilizers. They are easy to ionize, especially PEG, and if they are present, that is all you will see! You MUST AVOID DETERGENT for the vials you are using for mass spectrometry. COLORED TUBES AND TIPS: DO NOT USE IN ANY STEP. These materials often contain residual quantities of dyes that can be release into your sample. We provide some options "mass spec friendly" list. DO NOT SEAL TUBES WITH PARAFILM. They are a rich source of PEG contamination.

Miscellaneous

A COUPLE OF VERY IMPORTANT THINGS TO AVOID OTHER CONTAMINANTS 1. Any sample manipulation should be done in a BSC, laminar flow hood, or by using mask and disposable surgical cap. 2. Always wear nitrile (not latex) gloves. 3. Wear a lab coat and make sure there is no gap between your coat sleeve and the gloves (lab tape and rubber bands work well). 4. All the bottles and containers that are purchased for MS metabolomics should be dedicated for MS metabolomics use only. Do not wash these with detergent. Do not share with others in your lab for non-MS purposes.

Reagents

RECOMMENDED REAGENTS AND MATERIALS

Vendor Cat# Item Brand FisherSci PI85188 Acetonitrile LC/MS Grade thermo Chemical Thermo-Pierce FisherSci 14-432-22 Falcon 50mL Conical Centrifuge Tubes Fischer FisherSci 14-959-53A Falcon™ 15mL Conical Centrifuge Tubes Fischer FisherSci 19-177- 520 Gloves, Nitrile; Microflex XCEED; Latex free Xceed FisherSci 10-320-734 Low-Binding Rack Pipet Tips 10 uL Corning FisherSci 02-717-150 Low-Binding Rack Pipet Tips 1000 uL Fischer FisherSci 10-320-735 Low-Binding Rack Pipet Tips 200 uL Corning FisherSci A456-212 Methanol, Optima™ LC/MS Grade, Fisher Chemical Fischer FisherSci 13 688 654 Pipet Tips 1 to 10mL Fischer FisherSci 60-001-35 PROPANOL CHRMSLV LC-M 2.5LT Honeywell Riedel-de Haen FisherSci 02-681-320 Microcentrifuge Siliconized Low-retention 1.5 mL Fischer FisherSci 02-681-321 Microcentrifuge Siliconized Low-retention 2.0 mL Fischer FisherSci 600-30-79 Water with 0.1% Formic Acid, LC-MS and HPLC Honeywell Burdick &Jackson

SOLUTIONS

All solutions must be freshly prepared. Make sure you have enough solution (mobile phase and sample reconstitute) for all the samples, prior to your run. All reagents must be at least HPLC grade for MS experiments. For more information consult the list of recommended material. All these protocols provided are suggestions and provide good starting point. The results can vary according to sample. You can optimize the protocols for your experiment.

Extraction solution A = 100% Methanol containing one IS for positive mode and one for negative mode. Extraction solution B = 0.1% Formic Acid in Water (LC-MS grade) containing one IS for positive mode and one for negative mode. Extraction solution C = Methanol/Water (1:1) containing one IS for positive mode and one for negative mode. Extraction solution D = Dichloromethane/methanol (3:1) containing one IS for positive mode and one for negative mode. Extraction solution E = Chloroform/Methanol/Water (1:3:1 ratio) containing one IS for positive mode and one for negative mode. Extraction solution F = 80% Methanol containing one IS for positive mode and one for negative mode...

Internal Standards (could be stable isotope labelled) Mixture Components Positive mode Negative mode Expected mass ug/mL Expected mass ug/mL Caffeine 195.0882 1.5 Not applicable - Sulfadimethoxine (SDM) 311.0814 1 309.0658 1 Reserpine 609.2812 0.6 Not applicable - Leucine-Enkephalin (Leu-Enk) 556.2771 2.5 554.2615 2.5 4-Nitrobenzoic acid (NTA) Not applicable - 166.0146 10 Sulfaguanidine (SG) 215.0603 5 213.0446 5

Sample type Caffeine SDM Reserpine Leu-Enk NTA SG Human + (see note) + + (see note) - + + Rodent + (see note) + (see note) + (see note) - + + (see note) Mammalian cells + + + - + + Microbial cells + + + + + +

Study Design

Study design

Definition of technical and biological replicates depends on the model. „ For omics between 4-6 replicates is a good starting point for a pilot study. Depending on your study subject (animals, cells etc.) your replicate number should change. Cultured cells n=5-10, for animal models=7-12, for plant models n=7-12, for controlled clinical trials n=12-25, uncontrolled clinical trials n=50-100, epidemiology studies n=100-1000. (these numbers are taken from “Waters Metabolomics workshop study design data analysis”, they might vary)

Quality Control (QC)

The purpose of QCs is to monitor the performance of metabolomics workflows (extraction and acquisition). You should prepare QCs before extraction by mixing equal volumes from each sample and continue same protocol of extraction with all your samples and QC, together. Preparation of the QCs should follow the same protocol performed for samples including number of freeze-thaw cycles. If it is not possible to create a pooled QC sample due to limited sample amounts commercially available QC sample may be used (ex: human serum)

Blank

You should do the same extraction protocol with only solutions and tubes you have been using for the other samples. That should not contain any sample or QC. This will eliminate false discoveries.

Internal standard(s)

Added in first step of sample extraction, generally at the extraction solution. „Please select your internal standard wisely prior to your extraction, it should be exogenous and not present in your subject. Prefer labelled sample.

Randomization

Randomization of sample preparation order to avoid systematic bias. In an ‘omics’ experiment, a batch is defined as a set of samples that have been extracted as well as measured in one uninterrupted sequence. Most often, batch effects in large untargeted metabolomics experiments are almost unavoidable and hence the goal of batch correction is to remove these between-batch and within-batch effects. Accounting for these batch effects ensures that the measurements across all batches are directly comparable. One of the key steps for mitigating systematic bias is ensuring randomization of samples and sample run order. Ideally, randomization of samples should be implemented right at the start of the experiment (from sample handling, processing) till the conclusion of the run on the instrument. The key goal of sample randomization is to achieve a COMPLETELY RANDOMIZED AND BALANCED sample set.

Chapters and Notes

Chapter I – Sample Label and Excel Spreadsheet if you are doing Colloboration MS analysis are very expensive labor we should let the colloborators know about the details of the cost before starting the project! - All samples MUST to be labelled or bar-coded and an Excel sheet should be provided to the colloborators before MS analysis. - Label must be made on both, lids and sides of the tubes, with water resistant markers or bar code tags. - The labelling system should be unique and contain PI initials (two letters), in charge student initials (two letters) and numbered form 001-1000…. - All the tubes MUST to be in a Microtube Storage Boxes - All Storage boxes MUST have a tag in the lid (Figure 2) containing all important information about samples. Ex: PI: Robert Powers, Student: John Doe, Samples 1-10 Label as F RPJD001, RPJD002 ….RPJD010

Project: Yeast metabolome PI: Robert Powers Student: John Doe Sample type: Yeast cell pellet Collected Date: 01/31/2019 Delivered Date: Analysis: MS untargeted metabolomics Project cost: Comment:Samples defrosted 1x NOTE: IT IS ESSENTAIL TO SEND AN EXCEL SPREADSHEET THAT HAS DETAILS OF ALL SAMPLES AND DETAILED TOTAL COST OF THE PROJECT TO THE COLLOBORATOR. This is to guarantee we have organized appropriate storage facility well in advance of receiving samples and to avoid any kind of financial conflict with the colloborator. (Reach out Aline De Lima Liete a.lima@unl.edu for the details of the excel spreadsheet).

Chapter II – Sample collection, storage and transport Please reach out either Isin isin-tuna.sakallioglu@huskers.unl.edu or Aline De Lima Liete a.lima@unl.edu for this chapter.

Chapter III – Metabolites extraction Please reach out either Isin isin-tuna.sakallioglu@huskers.unl.edu or Aline De Lima Liete a.lima@unl.edu for this chapter.

Appendix Please reach out Amith Maroli amith.maroli@unl.edu for the following informations: I. Randomization II. Bar-coding protocol (very important step in storage of the samples, learn it well!)