title: Quantitative multiple reaction monitoring analysis of synaptic proteins from human brain.
abstract: BACKGROUND
  The recent introduction of multiple reaction monitoring to proteomics research has allowed many researchers to apply this technique to study human diseases.
  
  
  METHODS
  Here we combine subcellular fractionation of human autopsy brain with label-free multiple reaction monitoring to quantitatively analyse proteins in synapses. The protein enolase, from Streptococcus pyogenes serotype M6, which is sufficiently different from human proteins, was spiked into the sample mixture prior to trypsin digestion and used as an internal standard across samples.
  
  
  RESULTS
  Three synaptic proteins and an internal standard analysed with four injections over four consecutive days gave consistent differences with a coefficient of variation of <4%. Consistent retention time was recorded across the replicates. Comparison with existing methods: Previously, multiple reaction monitoring analysis has been utilized to study human autopsy and animal tissues. Utilizing the synaptosomal fraction prior to analysis reduced sample complexity and allowed the enriched synaptic proteins to be quantitatively assessed in a highly reproducible manner, without the need for expensive fluorescent labels and synthetic peptides.
  
  
  CONCLUSIONS
  Protein expression can be measured with accuracy using label-free multiple reaction monitoring mass spectrometry in relatively complex human brain samples. Synaptic functions are critical for neuronal communication and function, and synapse dysfunction underlies many neurodegenerative diseases, including Alzheimer's disease. This method can be applied to study a range of brain disorders.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Australia. Electronic address: rachel.chang@uqconnect.edu.au.
authors.forename: Rachel Yoon Kyung
authors.initials: RY
authors.lastname: Chang
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Australia. Electronic address: n.etheridge@uq.edu.au.
authors.forename: Naomi
authors.initials: N
authors.lastname: Etheridge
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Australia. Electronic address: p.dodd@uq.edu.au.
authors.forename: Peter
authors.initials: P
authors.lastname: Dodd
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Australia. Electronic address: a.nouwens@uq.edu.au.
authors.forename: Amanda
authors.initials: A
authors.lastname: Nouwens
mesh_terms: D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism; D050863:Synaptotagmin I / Q000737:chemistry / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010455:Peptides; D050863:Synaptotagmin I; C533522:TUBA1A protein, human; D014404:Tubulin; D002402:Cathepsin D; D010751:Phosphopyruvate Hydratase
keywords: Alzheimer's disease; Human autopsy brain; Neurodegeneration; Sample enrichment; Synapse