Diagnosis and Classification of Amyloidosis in Abdominal Subcutaneous Fat Aspiration Specimens Using Mass Spectrometry Based Proteomics

Abstract

Abdominal subcutaneous fat aspiration is one of the most practical, sensitive and specific methods for the diagnosis of systemic amyloidosis. One limitation of this method, compared to more invasive tissue biopsy based approaches, remains the technical difficulties in further classification of the amyloidosis as commonly used methods, such as immunohistochemistry, are not readily applicable to fat aspiration specimens. To overcome these difficulties we developed a method using nano-flow liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) that could identify amyloid subtypes in freshly obtained Congo Red positive fat aspirate specimens with great accuracy.Abdominal subcutaneous fat aspirate specimens were obtained from 73 patients with clinical suspicion for systemic amyloidosis. One half of the specimen was stained with Congo red and used for diagnosis of amyloidosis and the other half was processed and enzyme digested for LC-MS/MS analysis. The resulting LC-MS/MS data was correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Scaffold (Mascot, Sequest, and X!Tandem search algorithms). Peptide identifications were accepted if they could be established at greater than 90.0% probability and protein identifications were accepted if they could be established at greater than 90.0% probability and contain at least 2 identified spectra. The identified proteins were subsequently examined for the presence or absence of amyloid related peptides. Of the 73 cases studied, 41 were positive for Congo red consistent with systemic amyloidosis. In Congo red positive cases, LC-MS/MS peptide profiles consistent with AL-lambda (28/31), AL-kappa (6/7), and ATTR (2/3) were observed. Only one case in the Congo red negative control group (31/32) gave a kappa light chain profile which was attributed to a high level of kappa in the serum (285 mg/dL). Of the 35 out of 41 cases of systemic amyloidosis successfully classified by LC-MS/MS, additional clinical and pathology data validating the amyloid type was available. In each of these cases the MS/MS results accurately predicted the amyloid type.In conclusion, LC-MS/MS proteomic analysis of abdominal subcutaneous fat aspiration specimens involved by amyloidosis provides a highly specific (97% specificity) and sensitive (>85% sensitivity) method for diagnosis and classification of amyloidosis. The method is rapid and readily applicable in a clinical setting and will greatly improve the clinical management of amyloidosis patients.