title: Targeting LAMP2 in human cerebrospinal fluid with a combination of immunopurification and high resolution parallel reaction monitoring mass spectrometry.
abstract: BACKGROUND
  Alzheimer's disease is the most common form of dementia. An increasing body of evidence suggests that endo-lysosomal dysfunction is a pathogenic mechanism of Alzheimer's disease. Thus there is a potential for proteins involved in the normal function of endo-lysosomal vesicles to act as biomarkers of disease. Herein we focused on the lysosomal protein LAMP2 that is involved in chaperone mediated autophagy.
  
  
  RESULTS
  Using a combination of immunoprecipitation, digestion and nano-liquid chromatography tandem mass spectrometry we targeted and identified six tryptic LAMP2 peptides in human cerebrospinal fluid. Employing the identified proteotypic tryptic peptides a hybrid immunoprecipitation high resolution parallel reaction monitoring mass spectrometric method was developed for the relative quantitation of LAMP2. The method was evaluated in a number of experiments which defined the overall methodological as well as the analytical micro-liquid chromatography mass spectrometric intra- and inter-day variability. We identified an overall methodological peptide dependent intra-day variability of 8-16 %. The inter-day experiments showed similar results. The analytical contribution to the variation was minor with a coefficient of variation of 0.5-2.1 %, depending on the peptide. Using the developed method, with defined and limited variability, we report increased cerebrospinal fluid levels of three LAMP2 peptides in Alzheimer's disease subjects (n = 14), as compared to non-Alzheimer's disease controls (n = 14).
  
  
  CONCLUSIONS
  Altered LAMP2 levels in cerebrospinal fluid may indicate endo-lysosomal dysfunction in Alzheimer's disease. However, further studies in larger cohorts comprised of well-defined patient materials are required. We here present a tool which can be used for exploring the relevance of the level of LAMP2 as a potential measure of lysosomal dysfunction in Alzheimer's disease or other neurodegenerative diseases.
authors.affiliation: Department of Psychiatry and Neurochemistry, Institution of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital at Mölndal, University of Gothenburg, House V3, 431 80 Mölndal, Sweden.
authors.forename: Simon
authors.initials: S
authors.lastname: Sjödin
authors.affiliation: Department of Psychiatry and Neurochemistry, Institution of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital at Mölndal, University of Gothenburg, House V3, 431 80 Mölndal, Sweden.
authors.forename: Annika
authors.initials: A
authors.lastname: Öhrfelt
authors.affiliation: Department of Psychiatry and Neurochemistry, Institution of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital at Mölndal, University of Gothenburg, House V3, 431 80 Mölndal, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Brinkmalm
authors.affiliation: Department of Psychiatry and Neurochemistry, Institution of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital at Mölndal, University of Gothenburg, House V3, 431 80 Mölndal, Sweden ; UCL Institute of Neurology, University College London, London, UK.
authors.forename: Henrik
authors.initials: H
authors.lastname: Zetterberg
authors.affiliation: Department of Psychiatry and Neurochemistry, Institution of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital at Mölndal, University of Gothenburg, House V3, 431 80 Mölndal, Sweden.
authors.forename: Kaj
authors.initials: K
authors.lastname: Blennow
authors.affiliation: Department of Psychiatry and Neurochemistry, Institution of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital at Mölndal, University of Gothenburg, House V3, 431 80 Mölndal, Sweden.
authors.forename: Ann
authors.initials: A
authors.lastname: Brinkmalm
keywords: Alzheimer’s disease; Biomarker; Cerebrospinal fluid; Endo-lysosomal dysfunction; Immunoprecipitation; LAMP2; Neurodegenerative diseases; Parallel reaction monitoring