Clinical amyloid subtyping from FFPE biopsies using a rapid preparation workflow and high-flow LC-Q Exactive MS
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Updated: Nov 8, 2026
Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain
Published on: April 28, 2022
Naga Veera Yerra1, Srinivas V S Chakravartula2, Coryn Gonis1
1Clinical Chemistry Service, Department of Pathology, Molecular and Cell-Based Medicine, The Mount Sinai Hospital, New York, NY 10029, USA.
Abstract:
Accurate amyloid protein typing is essential for diagnosis and treatment selection in patients with amyloidosis. Laser microdissection (LMD) followed by liquid chromatography-mass spectrometry provides characterization of amyloid deposits but may require prolonged sample preparation and analysis. We developed and evaluated a high-flow LC-MS/MS workflow for amyloid subtyping from formalin-fixed, paraffin-embedded (FFPE) tissue. Congo red-positive deposits were isolated by LMD from FFPE tissue and subjected to heat-assisted protein extraction and short-duration Trypsin/Lys-C digestion (3 h). Peptides were analyzed by high-flow LC-MS/MS using a Q Exactive Orbitrap with a 28-min gradient. Amyloid subtypes were identified on predefined protein sequence coverage, peptide-spectrum match, unique peptide, and amyloid-associated protein criteria. Analytical performance was evaluated using protein standards, LMD tissue dilution studies, precision, interference, stability, and blinded clinical specimens compared with a reference laboratory method. Sample preparation was completed within 6 h, enabling same-day analysis. A minimum LMD tissue area of >270,000 μm2 was established for reliable clinical interpretation. In blind clinical specimens, amyloid subtype assignment was concordant with Mayo Clinic reference laboratory results in 20 of 20 cases. Intra- and inter-run studies demonstrated stable chromatographic performance and reproducible amyloid protein identification. Increasing concentrations of albumin and hemoglobin reduced amyloid protein recovery, defining an important preanalytical limitation of the assay. This rapid high-flow LC-MS/MS workflow enables same-day amyloid protein subtyping from LMD FFPE tissue. The combination of standardized tissue input, short-duration digestion, short-gradient LC-MS/MS, and predefined protein identification criteria supports implementation of this approach in clinical laboratories.