Developing a mass spectrometric assay to measure granulin peptides in CSF for progranulin‐associated frontotemporal dementia

Abstract

AbstractBackgroundPathogenic mutations in the progranulin gene (GRN) are a key cause of frontotemporal dementia (FTD), inducing a reduced biofluid concentration of the progranulin protein (PGRN). PGRN is a cysteine‐rich glycoprotein with essential roles in inflammation and lysosomal function, made up of 7 granulin peptides and 1 paragranulin. The role of these peptides is unclear, but existing data suggests they may have contradictory roles to full‐length PGRN. With the development of numerous clinical trials aiming to treat progranulin‐associated FTD (FTD‐GRN) by increasing full‐length PGRN, it is important to establish effective outcome measures to assess treatment success and further our understanding of PGRN’s biology. Here, we aimed to develop an assay to quantify granulin peptides in cerebrospinal fluid (CSF) and determine whether they contribute to the pathology of FTD‐GRN.MethodBased on previously published explorative data of endogenous CSF peptides, 12 peptides spanning the progranulin sequence, were selected for the development of targeted assays using a quadrupole Orbitrap hybrid mass spectrometer (Fusion Tribrid, Thermo). An analytical protocol was optimised involving reduction, alkylation, molecular weight cut‐off filtration and solid phase extraction, and using isotope labelled heavy standards for quantification. Additionally, tandem mass tag (TMT) proteomics was used to analyse tryptic peptides spanning granulin and paragranulin sequence regions in 248 CSF samples from the Genetic FTD initiative (GENFI) including 56 GRN mutation carriers and 76 mutation‐negative controls.ResultPreliminary results reveal the presence of three endogenous peptides in CSF, which based on sequence matching, likely represent granulin 6 and 7 alongside the paragranulin peptide. TMT results showed significantly reduced relative peptide intensity across the granulin regions in GRN carriers CSF compared to controls (p<0.0001), but no significant difference in the paragranulin region (p>0.33).ConclusionThese findings indicate that two granulins and paragranulin are quantifiable in CSF and may have key roles in progranulin biology and potentially FTD pathology. This is supported by TMT results of differential CSF paragranulin levels compared to granulins. Continuing work will quantify CSF granulin concentrations in the GENFI cohort to assess whether these peptides have key roles in FTD‐GRN’s underlying biology and as potential outcome measures in trials.