Amyloidosis diagnosis by mass spectrometry

Jakob C. Albrethsen1, Morten Salomo2, Anders H. Johnsen3,*

1Dept. Clinical Biochemistry, 2Dept. Hemathology, 3Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark

Introduction: Amyloidose is a disorder characterized by protein deposition in various tissues and organs due to change in the proteins secondary structure. The accumulation of these insoluble aggregates leads to dysfunction of the affected organ, especially kidney, heart, gut, lungs and nerves – untreated with potentially lethal outcome. Treatment of amyloidosis depends on the type of involved protein. More than 20 different proteins causing amyloidosis are known, immunoglobulin light chains produced by monoclonal plasma cells being the most common.

Objectives: In 80% of systemic amyloidosis cases, protein deposits can be found in subcutaneous abdominal fat, a specimen that is easily obtainable by needle biopsy. However, traditional examination based on histochemistry is often unreliable and novel MS-based proteomics methods have been suggested (e.g.*). The purpose of our work is to establish such a method.

Methods: Fat tissue biopsies are collected by needle-aspiration. Total protein is extracted by repeated sonication and shaking in lysis buffer (8M Urea, CHAPS, protease inhibitors). The samples (5ug protein) are dialysed, and subjected to SDS-PAGE and in-gel digestion (6 pieces of the gel). The tryptic peptides are extracted from the gel and analysed by nanoLC-MS using a 180 min gradient (Ultimate3000 LC system (Thermo) coupled with a MicrO-TOF QII MS instrument (Bruker)). The results are evaluated by semi-quantitative label-free analysis using the Mascot Distiller software (Matrix Science).

Results: The method confidently identified light chain kappa/lambda, and serum amyloid A in patients with AL- and AA-amyloidosis, respectively (as well as ~300 other proteins).

Conclusion: Our preliminary data support that semi-quantitative label-free LCMS can be used in typing of amyloid deposits. However, only a few control samples and patients have been analysed so far. Thus further work is warranted to validate this promising method in our lab.

*Brambilla F et al. Blood 2012; 119: 1844-7

Keywords: Biomarker development, Method, Multiple organs