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).Conclusion: 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.

Proteomic studies of cerebrospinal fluid biomarkers of Alzheimer’s disease: an update

“…The levels of all three peptides were reported to increase in AD compared to controls. LAMP2 did not correlate with any of the AD core biomarkers [42]. [43].…”

“…Hence, proteins involved in the normal function of endo-lysosomal vesicles could be tentative biomarkers of AD. The lysosomal protein LAMP2 is involved in chaperone mediated autophagy and recently it was shown that the concentrations of three CSF LAMP2 peptides were increased in AD compared to non-AD controls [42]. Altered CSF LAMP2 concentrations may indicate endo-lysosomal dysfunction in AD, but further studies of larger cohorts also including patients with other neurodegenerative disorders are required.…”

A Parallel Reaction Monitoring Mass Spectrometric Method for Analysis of Potential CSF Biomarkers for Alzheimer's Disease

“…The approach has been successfully implemented previously both by our laboratory and others . When attempting this approach with an abundance difference of 10 3 or higher the low intensity peak reproducibility was reduced (data not shown) and the standard approach with separate isolations should be used.…”

Mass Spectrometric Analysis of Cerebrospinal Fluid Ubiquitin in Alzheimer's Disease and Parkinsonian Disorders

“…PRM offers highly accurate and selective measurements and can successfully be applied to the analysis of complex samples . With good precision, we have previously measured the lysosomal membrane protein LAMP2 in CSF using PRM . Furthermore, measurements using PRM have shown similar performance compared to SRM, however, with the advantage of a broader dynamic range …”