Targeting LAMP2 in human cerebrospinal fluid with a combination of immunopurification and high resolution parallel reaction monitoring mass spectrometry

Simon Sjödin, Annika Öhrfelt, Gunnar Brinkmalm, Henrik Zetterberg, Kaj Blennow, Ann Brinkmalm

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. 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). 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.