Rapid method to characterize mutations in transthyretin in cerebrospinal fluid from familial amyloidotic polyneuropathy patients by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Background: Familial amyloidotic polyneuropathy (FAP) type I, the most comm on dominantly inherited form of amyloidosis, is caused by a Val-to-Met poin t mutation at position 30 (Val(30)-->Met) in the protein transthyretin. Mas s spectrometric analysis can identify modification of proteins, such as poi nt mutations, acetylation, phosphorylation, sulfation, oxidation, and glyco sylation. Methods: Matrix-assisted laser desorption/ionization time-of-flight mass sp ectrometry (MALDI-TOF MS) spectra from cerebrospinal fluid (CSF) drawn from a patient with FAP were compared with CSF from controls. We also isolated transthyretin with a Centrisart molecular size cutoff filter and performed high-accuracy peptide mass mapping to localize the site of the amino acid s ubstitution (Val(30)-->Met). Results: Mass spectra of transthyretin were produced directly from human CS F as well as from CSF after a simple prepurification method without immunop recipitation. On-target tryptic digestion and MALDI-MS verified mass spectr ometric peak identification. The point mutation was still detectable in CSF after hepatic transplantation. Conclusions: It is possible to diagnose FAP by a rapid MALDI-TOF MS analysi s using only 100 mu L of CSF, with only 250 nL actually consumed on target. The approach may also be useful to monitor production of mutated transthyr etin by choroid plexus, especially after liver transplantation. (C) 2000 Am erican Association for Clinical Chemistry.