Matthew B O'Rourke1, 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: This paper, in conjunction with a work published earlier this year by O'Rourke et al., aims to provide a comprehensive set of protocols for the analysis of formalin-fixed paraffin-embedded (FFPE) tissue via matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) in a low-cost and highly repeatable and robust way, thereby allowing other research teams to begin their own IMS-centered avenues of research. METHODS: Samples of FFPE tissue were sectioned at 5 µm, water float mounted to specially prepared ITO glass slides and then dilipidated in a graded alcohol series. Tissue sections were then antigen retrieved under pressure in 20 mmol Tris-HCl (pH 8.8), coated with trypsin and digested O/N at 37°C. Samples were then sublimated with matrix to a final coverage of 0.2 mg/cm(2), recrystallised at 37°C with 50:50 acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) for 1 h and analysed with a MALDI TOF/TOF mass spectrometer. RESULTS: Serial sections were imaged, revealing little to no variation with regards to image quality and corresponding spectra. We have also attempted to describe the processes that govern the various aspects of this protocol with respect to each step necessary to ensure reproducibility. CONCLUSIONS: We are confident that this protocol in conjunction with the work published earlier by O'Rourke et al. provides the basis for a repeatable and robust protocol for the analysis of tissues from various sources via MALDI-IMS. The descriptions of key steps within allows for easy adoption of the protocol while allowing for desired modifications to be performed with minimal yet intuitive adjustment.
RATIONALE: This paper, in conjunction with a work published earlier this year by O'Rourke et al., aims to provide a comprehensive set of protocols for the analysis of formalin-fixed paraffin-embedded (FFPE) tissue via matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) in a low-cost and highly repeatable and robust way, thereby allowing other research teams to begin their own IMS-centered avenues of research. METHODS: Samples of FFPE tissue were sectioned at 5 µm, water float mounted to specially prepared ITO glass slides and then dilipidated in a graded alcohol series. Tissue sections were then antigen retrieved under pressure in 20 mmol Tris-HCl (pH 8.8), coated with trypsin and digested O/N at 37°C. Samples were then sublimated with matrix to a final coverage of 0.2 mg/cm(2), recrystallised at 37°C with 50:50 acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) for 1 h and analysed with a MALDI TOF/TOF mass spectrometer. RESULTS: Serial sections were imaged, revealing little to no variation with regards to image quality and corresponding spectra. We have also attempted to describe the processes that govern the various aspects of this protocol with respect to each step necessary to ensure reproducibility. CONCLUSIONS: We are confident that this protocol in conjunction with the work published earlier by O'Rourke et al. provides the basis for a repeatable and robust protocol for the analysis of tissues from various sources via MALDI-IMS. The descriptions of key steps within allows for easy adoption of the protocol while allowing for desired modifications to be performed with minimal yet intuitive adjustment.
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