title: Proteomic MALDI-TOF/TOF-IMS examination of peptide expression in the formalin fixed brainstem and changes in sudden infant death syndrome infants.
abstract: Matrix assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) has not previously been utilised to examine sudden infant death syndrome (SIDS). This study aimed to optimise MALDI IMS for use on archived formalin-fixed-paraffin-embedded human infant medulla tissue (n=6, controls; n=6, SIDS) to evaluate differences between multiple nuclei of the medulla by using high resolution IMS. Profiles were compared between SIDS and age/sex matched controls. LC-MALDI identified 55 proteins based on 321 peptides across all samples; 286 peaks were found using IMS, corresponding to these 55 proteins that were directly compared between controls and SIDS. Control samples were used to identify common peptides for neuronal/non-neuronal structures allowing identification of medullary regions. In SIDS, abnormal expression patterns of 41 peptides (p≤0.05) corresponding to 9 proteins were observed; these changes were confirmed with immunohistochemistry. The protein abnormalities varied amongst nuclei, with the majority of variations in the raphe nuclei, hypoglossal and pyramids. The abnormal proteins are not related to a previously identified neurological disease pathway but consist of developmental neuronal/glial/axonal growth, cell metabolism, cyto-architecture and apoptosis components. This suggests that SIDS infants have abnormal neurological development in the raphe nuclei, hypoglossal and pyramids of the brainstem, which may contribute to the pathogenesis of SIDS.
  
  
  CONCLUSIONS
  This study is the first to perform an imaging mass spectrometry investigation in the human brainstem and also within sudden infant death syndrome (SIDS). LC MALDI and MALDI IMS identified 55 proteins based on 285 peptides in both control and SIDS tissue; with abnormal expression patterns present for 41/285 and 9/55 proteins in SIDS using IMS. The abnormal proteins are critical for neurological development; with the impairment supporting the hypothesis that SIDS may be due to delayed neurological maturation. The brainstem regions mostly affected included the raphe nuclei, hypoglossal and pyramids. This study highlights that basic cyto-architectural proteins are affected in SIDS and that abnormal expression of these proteins in other CNS disorders should be examined.
  LC MALDI and MALDI IMS identified 55 proteins based on 285 peptides in both control and SIDS tissue. Abnormal expression patterns were present for 41/285 and 9/55 proteins in SIDS using IMS. Brainstem regions mostly affected included the raphe nuclei, hypoglossal and pyramids.
authors.affiliation: Department of Medicine, Central Clinical School, University of Sydney, NSW, Australia; BOSCH Institute of Biomedical Research, University of Sydney, NSW, Australia.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Hunt
authors.affiliation: Hormones and Cancer Division, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, NSW, Australia.
authors.forename: Leo
authors.initials: L
authors.lastname: Phillips
authors.affiliation: Department of Medicine, Central Clinical School, University of Sydney, NSW, Australia; BOSCH Institute of Biomedical Research, University of Sydney, NSW, Australia; The Children's Hospital, Westmead, NSW 2145, Australia.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Waters
authors.affiliation: Department of Medicine, Central Clinical School, University of Sydney, NSW, Australia; BOSCH Institute of Biomedical Research, University of Sydney, NSW, Australia; The Children's Hospital, Westmead, NSW 2145, Australia. Electronic address: rita.machaalani@sydney.edu.au.
authors.forename: Rita
authors.initials: R
authors.lastname: Machaalani
mesh_terms: D001933:Brain Stem / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D007223:Infant; D008297:Male; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D010455:Peptides / Q000378:metabolism*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013398:Sudden Infant Death; D016707:Tissue Fixation
chemical_list: D009419:Nerve Tissue Proteins; D010455:Peptides; D005557:Formaldehyde
keywords: Brain; Human; Imaging mass spectrometry; Immunohistochemistry; Paraffin-embedded; SIDS