Matthew B O'Rourke1, Benjamin B A Raymond2, Steven P Djordjevic2, Matthew P Padula1. 1. Proteomics Core Facility, University of Technology Sydney, Cnr Harris and Thomas St, Ultimo, NSW, 2007, Australia. 2. The iThree Institute, University of Technology Sydney, Cnr Harris and Thomas St, Ultimo, NSW, 2007, Australia.
Abstract
RATIONALE: There are currently multiple methods available for the preparation of fresh frozen tissue samples for analysis via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS). Although these methods report excellent results, many are expensive automated approaches. With no published attempt to standardise less expensive manual processes, our work aims to provide a robust and repeatable method of sample preparation for MALDI-TOF-IMS that is applicable to a variety of tissue types, well explained, simple and cost effective. METHODS: Fresh frozen tissue was sectioned at 12 µm and mounted onto liquid nitrocellulose coated slides, washed in a graded alcohol series and then mounted into a modified sublimation apparatus. Matrix is deposited onto the slide to achieve a desired coating of 0.2 mg/cm(2). Once coated, the slide is mounted into a custom-built vapor chamber and recrystallised with 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) for 1 h at 37°C. The slide is then analysed using MALDI-IMS. RESULTS: We have successfully implemented this method for a host of tissue samples, including brain, liver, kidney and heart, with no variation in relative spectra or processing method required. When the protocol is followed correctly, sublimations and recrystallisations are highly predictable with limited variation between samples and a very low failure rate. Additional apparatuses can be easily constructed by following the included instructions, that perform as per specifications with no variation. CONCLUSIONS: We believe that we have described a complete protocol for MALDI-IMS that is easy to use and highly reproducible. The lack of expensive commercially available equipment makes this process very cheap with a relatively low initial outlay and our hope is that more laboratories will begin IMS-based avenues of research based on the work we have performed.
RATIONALE: There are currently multiple methods available for the preparation of fresh frozen tissue samples for analysis via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS). Although these methods report excellent results, many are expensive automated approaches. With no published attempt to standardise less expensive manual processes, our work aims to provide a robust and repeatable method of sample preparation for MALDI-TOF-IMS that is applicable to a variety of tissue types, well explained, simple and cost effective. METHODS: Fresh frozen tissue was sectioned at 12 µm and mounted onto liquid nitrocellulose coated slides, washed in a graded alcohol series and then mounted into a modified sublimation apparatus. Matrix is deposited onto the slide to achieve a desired coating of 0.2 mg/cm(2). Once coated, the slide is mounted into a custom-built vapor chamber and recrystallised with 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) for 1 h at 37°C. The slide is then analysed using MALDI-IMS. RESULTS: We have successfully implemented this method for a host of tissue samples, including brain, liver, kidney and heart, with no variation in relative spectra or processing method required. When the protocol is followed correctly, sublimations and recrystallisations are highly predictable with limited variation between samples and a very low failure rate. Additional apparatuses can be easily constructed by following the included instructions, that perform as per specifications with no variation. CONCLUSIONS: We believe that we have described a complete protocol for MALDI-IMS that is easy to use and highly reproducible. The lack of expensive commercially available equipment makes this process very cheap with a relatively low initial outlay and our hope is that more laboratories will begin IMS-based avenues of research based on the work we have performed.
Authors: Matthew B O'Rourke; Benjamin B A Raymond; Steven P Djordjevic; Matthew P Padula Journal: J Am Soc Mass Spectrom Date: 2018-01-08 Impact factor: 3.109
Authors: Matthew B O'Rourke; Matthew P Padula; Caine Smith; Priscilla Youssef; Stuart Cordwell; Paul Witting; Greg Sutherland; Ben Crossett Journal: J Vis Exp Date: 2018-01-16 Impact factor: 1.355
Authors: Matthew B O'Rourke; Ben R Roediger; Christopher J Jolly; Ben Crossett; Matthew P Padula; Phillip M Hansbro Journal: Proteomes Date: 2022-09-13
Authors: Heeva Baharlou; Nicolas P Canete; Anthony L Cunningham; Andrew N Harman; Ellis Patrick Journal: Front Immunol Date: 2019-11-14 Impact factor: 7.561