Apr 10, 2023
High Spatial Resolution MALDI Imaging Mass Spectrometry Data Acquisition
- Katerina V Djambazova1,
- Angela R.S. Kruse1,
- Jamie Allen1,
- allison.b.esselman1,
- Madeline E. Colley1,
- David Anderson1,
- Ali Zahraei1,
- Olof Isberg1,
- Melissa Farrow1,
- Jeff Spraggins1,
- Martin Dufrense1
- 1Vanderbilt University
- VU Biomolecular Multimodal Imaging Center / Spraggins Research GroupTech. support email: jeff.spraggins@vanderbilte.du
Protocol Citation: Katerina V Djambazova, Angela R.S. Kruse, Jamie Allen, allison.b.esselman, Madeline E. Colley, David Anderson, Ali Zahraei, Olof Isberg, Melissa Farrow, Jeff Spraggins, Martin Dufrense 2023. High Spatial Resolution MALDI Imaging Mass Spectrometry Data Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpq99l5b/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: February 08, 2023
Last Modified: October 18, 2023
Protocol Integer ID: 76683
Keywords: MALDI IMS, Lipid Imaging
Abstract
This protocol provides the workflow and instrument parameters for setting up a MALDI imaging mass spectrometry experiment on a Bruker timsTOF Flex instrument. This protocol is intended for 10µm spatial resolution imaging of lipids (m/z 400 - 2000) in positive and negative ionization mode data acquisition in qTOF mode of operation.
Materials
Agilent ESI-L Tune Mix (Agilent Technologies, Santa Clara, CA)
Place slides in a MTP 2-slide holder. Scan slides using flatbed scanner, allowing for sufficient contrast to visualize the tissue boundary. Ensure the slides have fiducials.
Note
Fiducials are markings on the slide that can be used as reference points for teaching the slides (Step 4).
Load the 2-slide holder in the instrument. Open timsControl software and select appropriate MALDI IMS data acquisition method. Generate target profile by ensuring "Use Target Profile" is enabled. Check the height at a few different locations on the slide and ensure the difference is <10 µm.
Note
Example Positive/Negative Ionization Mode Parameters for 10µm MALDI IMS:
m/z 400 - 2000
Laser Power: 35 - 45%, Frequency: 10 000 Hz
Shots: 100 - 150 per pixel
Spot size: 10 µm with Beam scanning function disabled
Source temperature: ~50 oC
Calibrate the TOF with an external calibration using red phosphorus. Proceed if all calibration points are <1ppm standard deviation error and the calibration score is >99%.
Note
Red phosphorus is commonly used to perform calibration, however Agilent ESI-L Tune Mix can also be used for electrospray ionization.
List of Red Phosphorus Mass Calibration Peaks
| A | B | |
| Positive mode | Negative mode | |
| 278.7633 | 278.7644 | |
| 402.6583 | 402.6594 | |
| 464.6058 | 464.6069 | |
| 526.5534 | 526.5545 | |
| 650.4484 | 650.4495 | |
| 774.3435 | 774.3445 | |
| 898.2385 | 898.2396 | |
| 960.1860 | 960.1870 | |
| 1022.1335 | 1022.1346 | |
| 1146.0286 | 1146.0297 | |
| 1393.8187 | 1393.8198 | |
| 1517.7137 | 1517.7148 | |
| 1641.6088 | 1641.6099 | |
| 1765.5038 | 1765.5049 | |
| 1889.3989 | 1889.4000 |
Use the FlexImaging software to load the optical image of the slides. Teach the target position with three teaching points. Check the accuracy of the teaching by moving the stage to a fourth location.
Select desired tissue measurement region. Start data acquisition in the FlexImaging software.