Diagnostic application of proteomics: testing a new sample preparation for the typing of amyloid proteins in biopsies

Amyloidosis refers to a group of heterogeneous clinical conditions caused by the deposition of misfolded proteins in the extracellular matrix of tissues and organs. A key feature of these proteins is their tendency to form antiparallel beta-pleated sheets, which then become non-branching fibrils resistant to degradation. These fibrils cause mechanical damage and oxidative stress in organs like the heart, liver, kidneys, nervous system, and gastrointestinal tract. Τhe gold standard for diagnosing amyloidosis is Congo red staining of sections from formalin fixed paraffin embedded biopsies, showing amyloid deposits as green birefringent areas under polarized light. More than 40 precursor proteins are known to misfold and self-assemble as amyloids. Therefore, upon histological confirmation of the amyloid deposits, the identity of the amyloidogenic (precursor) protein must be determined, given that several amyloidoses have disease-specific therapies. This is called amyloid typing. Mass spectrometry-based bottom-up proteomic analysis of laser microdissected biopsy areas is currently the most reliable method to type amyloid. But sample preparation, as developed 10-15 years ago, is somewhat tedious as the fibrils are not easily lysed. We conducted experiments to compare the state-of-the-art sample preparation used for proteomic typing of amyloid to a simpler procedure inspired from the latest developments in other domains of proteomics. More specifically, we compared a protocol involving lysis with TrisBase, EDTA, Zwittergent (TEZ) buffer to a more user-friendly protocol featuring lysis with trifluoroacetic acid (TFA). Tryptic peptides were analysed on a timsTOF fleX (Bruker) using Data Independent Acquisition - Parallel Accumulation–Serial Fragmentation (DIA-PASEF). Raw data were processed with the DIA-NN library-free software. This study discusses the differences between the two methods in terms of 1) practicalities, cost and time, 2) number of protein groups detected and overlapping profiles, 3) capacity to conclude on the diagnosis.