title: Brain Region-Specific Dynamics of On-Tissue Protein Digestion Using MALDI Mass Spectrometry Imaging.
abstract: In mass spectrometry imaging (MSI), on-tissue proteolytic digestion is performed to access larger protein species and to assign protein identities through matching the detected peaks with those obtained by LC-MS/MS analyses of tissue extracts. The on-tissue proteolytic digestion also allows the analysis of proteins from formalin-fixed, paraffin-embedded tissues. For these reasons, on-tissue digestion-based MSI is frequently used in clinical investigations, for example, to determine changes in protein content and distribution associated with a disease. In this work, we sought to investigate the completeness and uniformity of the digestion in on-tissue digestion MSI. On the basis of an extensive experiment investigating three groups with varying incubation times: (i) 1.5 h, (ii) 3 h, and (iii) 18 h, we have found that longer incubation times improve the repeatability of the analyses. Furthermore, we discovered morphology-associated differences in the completeness of the proteolysis for short incubation times. These results support the notion that a more complete proteolysis allows better quantitation.
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.
authors.forename: Bram
authors.initials: B
authors.lastname: Heijs
authors.affiliation: Department of Neurology, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.|Department of Human Genetics, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.
authors.forename: Else A
authors.initials: EA
authors.lastname: Tolner
authors.affiliation: Department of Pathology, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.
authors.forename: Judith V M G
authors.initials: JV
authors.lastname: Bovée
authors.affiliation: Department of Neurology, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.|Department of Human Genetics, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.
authors.forename: Arn M J M
authors.initials: AM
authors.lastname: van den Maagdenberg
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.|Department of Pathology, Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.|Fondazione Pisana per la Scienza ONLUS , Pisa 56125, Italy.
authors.forename: Liam A
authors.initials: LA
authors.lastname: McDonnell
mesh_terms: D000818:Animals; D001921:Brain / Q000033:anatomy & histology*; D001923:Brain Chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D004676:Myelin Basic Protein / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014018:Tissue Distribution
chemical_list: D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins
keywords: MALDI mass spectrometry imaging; on-tissue digestion; proteolysis