TITLE: Bulk Untargeted LC-MS/MS Lipidomics
AUTHORS: Madeline E. Colley, Jamie Allen, Katerina V Djambazova, Angela  Kruse, melissa.a.farrow, Jeff Spraggins

[DESCRIPTION]
This protocol is the detailed methodology for extracting lipids from tissue, connecting a Waters UHPLC to a timsTOF mass spectrometer via contact closure and performing LC-MS/MS analysis.

[STEPS]
SECTION: Tissue Collection
2. Tissue is sectioned at 10 um width in a cryostat and (1-3) scrolls of tissue is placed into a thermo glass shell vial. 

Keep one empty vial with a random, known number on it without tissue inside for a sample blank.
 

SECTION: Preamble
1. Please reference the materials section for details on materials, consumables, equipment details and solvents used. In the protocol, they will be referred to in layman terms (ie "glass vial", "mobile phase A", etc.)

All pipettes used in this protocol are Drummond positive displacement with borosilicate tips. 
SECTION: Lipid Extraction by MTBE
3. Spike 5 uL Avanti Equisplash internal standard into your sample vial using the 1-25 uL Drummond pipette. 
SECTION: Lipid Extraction by MTBE
4. Add enough cold methanol to cover your tissue in the vial (400 uL for small sample amounts).
 

SECTION: Lipid Extraction by MTBE
5. Add 2 large and 3 medium sized stainless steel beads from a navy RINO homogenizer tube to your glass vials and vortex sample for 1 minute.
SECTION: Lipid Extraction by MTBE
6. Place samples on dry ice for 5 minutes.
SECTION: Lipid Extraction by MTBE
7. Place samples on wet ice for 5 minutes and sonicate on ice for 30 minutes.
SECTION: Lipid Extraction by MTBE
8. Repeat steps 6 and 7.
SECTION: Lipid Extraction by MTBE
9. Add 1600 uL cold tert-butyl methyl ether (MTBE) to the vial and vortex for 30 seconds.
SECTION: Lipid Extraction by MTBE
10. Sonicate for 30 minutes on ice.
SECTION: Lipid Extraction by MTBE
11. Add 400 uL of cold water for phase separation and vortex for 30 seconds. 
SECTION: Lipid Extraction by MTBE
12. Cetrifuge for 10 minutes at 100 xg
SECTION: Lipid Extraction by MTBE
13. Allow to separate further for 10 minutes on ice. 
SECTION: Lipid Extraction by MTBE
14. Collect 1500 uL of the top layer (MTBE) with the Drummond 250 uL pipette into an autosampler vial.
SECTION: Lipid Extraction by MTBE
15. For small sample amounts, repeat steps 9-14 an additional time and add the top layer to the same new autosampler vial from step 14.
SECTION: Lipid Extraction by MTBE
16. 
 

SECTION: Lipid Extraction by MTBE
17. Dry samples down to completion under nitrogen without heat using a sample concentrator.
SECTION: Lipid Extraction by MTBE
18. Store lipids in -80 C dried down. 
SECTION: Lipid Extraction by MTBE
19. Prior to analysis, resuspend sample in methanol to volume appropriate for analysis. For small sample amounts, we use 50 uL. For bulk samples, we use 500 uL. 
SECTION: Waters Premier Liquid Chromatography Preparation
20. Prepare your mobile phases using only UHPLC grade solvents and additives. Additives must be stored in a dry environment. 

Mobile Phase A: 60% ACN, 50% H2O, 10 mM ammonium formate, 0.1% formic acid
Mobile Phase BL 90% IPA, 10% ACN, 10 mM ammonium formate, 0.1% formic acid 

Degas solvents for 20 minutes by sonication.

 

SECTION: Waters Premier Liquid Chromatography Preparation
21. Perform system checks: 
1. Check solvent levels 
    SM = needle wash (IPA)
    SW = seal wash (90:10 ACN:H2O)
    PURGE = (90:10 ACN:H2O)
    A = desalting columns (H2O)
    B = desalting columns (ACN)
    C = Mobile phase A
    D = mobile phase B
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
27. 1. Open the relevant method for analysis 
    4D-lipidomics positive or negative with MS/MS Stepping

SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
28. Install appropriate ion source (ViP-hESI or Apollo)
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
29. Adjust all temperatures and flow rates appropriate for analysis and wait 30 minutes for TIMS Tunnel In pressure to stabilize.  
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
30. Attack PEEK tubing from syringe pump to the source inlet and flow Agilent low concentration tune mix and 180 uL/min. 
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
31. Calibrate mass and mobility: Adjust funnel pressure by selecting the lightning bolt icon in timsControl software (Display mobilogram in [V]. 
Adjust funnel pressure until the calibrant is at the appropriate [V]. 

Positive mode: m/z 622.029 should have 132.5 [V]
Negative mode: m/z 601.979 should have 118.5 [V]

Calibrate m/z dimension.

Repeat mobility calibration. 

Wait 20 minutes to assess drift. If system has not drifted in pressure, do not recalibrate. If system has drifted in pressure, recalibrate. 
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
32. Open the configuration editor software and select "WATERSRS232" method. 
This method has the timsTOF mass spectrometer as the instrument and the COM2 channel active for input with RS232. 

Set method to active.
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
33. Open Bruker HyStar software
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
34. Create matching sample tables in HyStar and MassLynx
SECTION: Waters Premier Liquid Chromatography Preparation
22. Restart system starting with turning on the sample manager near the mass spectrometer 
SECTION: Waters Premier Liquid Chromatography Preparation
23. Connect the contact closure RS232 cable from the back of the LC system to the back of the instrument computer. Connect ethernet cable to university internet.
SECTION: Waters Premier Liquid Chromatography Preparation
24. Open MassLynx and Console
SECTION: Waters Premier Liquid Chromatography Preparation
25. Change sample manager temp to 8C. Change column temperature to 55C.
SECTION: Waters Premier Liquid Chromatography Preparation
26. Connect your column (Waters Premier C18 CSH 2.1 mm x 100 mm) to the column manager and condition by running mobile phase a for 20 minutes.  
Ensure pressure delta goes to low double or single digits.
SECTION: Bruker timsTOF FleX Mass Spectrometer Preparation
35. Right click on the first line of your HyStar stample table and click "upload sample conditions"
SECTION: System Suitability Test (SST)
36. Prepare 5 serial dilutions of Avant Equisplash Mix and Sulfatide standard within the linear range of your sample concentration 
SECTION: System Suitability Test (SST)
37. Run all dilutions in RANDOM order with the same chromatography and mass spectrometer methods as your samples
SECTION: Sample Table Setup
40. Prepare your sample table so that you randomly sample across your QCs, blanks and samples. 
SECTION: System Suitability Test (SST)
38. Open data in skyline and ensure that retention times do not drift, area under the chromatographic curves are linear for all dilutions, and that all expected species with adducts are detected. 
SECTION: System Suitability Test (SST)
39. Ensure that your blank is "blank". 
SECTION: Sample Table Setup
41. You should have a solvent blank - in our case, methanol. 
A sample blank - a blank vial that endured the same extraction protocol as your samples.
A QC sample - take 5 uL off the top of all samples and add then to the same vial
SECTION: Data Analysis
42. Files are converted to IBF format using the converter provided in MS-DIAL 4.90
SECTION: Data Analysis
43. Data is input into MS-DIAL with the pooled reference sample being the "QC", any random blank being the "blank" and the samples being separated by their category (in our case by patient or condition).
SECTION: Data Analysis
44. Our MS Accuracy tolerance is 0.01 Da, with 0.05 MS2 tolerance. We allow for 500 minimum peak height. 
SECTION: Data Analysis
45. Once data is processed we filter by "Ref. matched" only and export this as our final list.