Tandem Mass Spectrometry-Based Amyloid Typing Using Manual Microdissection and Open-Source Data Processing.

OBJECTIVES

Standard implementations of amyloid typing by liquid chromatography-tandem mass spectrometry use capabilities unavailable to most clinical laboratories. To improve accessibility of this testing, we explored easier approaches to tissue sampling and data processing.

METHODS

We validated a typing method using manual sampling in place of laser microdissection, pairing the technique with a semiquantitative measure of sampling adequacy. In addition, we created an open-source data processing workflow (Crux Pipeline) for clinical users.

RESULTS

Cases of amyloidosis spanning the major types were distinguishable with 100% specificity using measurements of individual amyloidogenic proteins or in combination with the ratio of λ and κ constant regions. Crux Pipeline allowed for rapid, batched data processing, integrating the steps of peptide identification, statistical confidence estimation, and label-free protein quantification.

CONCLUSIONS

Accurate mass spectrometry-based amyloid typing is possible without laser microdissection. To facilitate entry into solid tissue proteomics, newcomers can leverage manual sampling approaches in combination with Crux Pipeline and related tools.