Shotgun‐proteomics‐based clinical testing for diagnosis and classification of amyloidosis

Abstract

Shotgun proteomics technology has matured in the research laboratories and is poised to enter clinical laboratories. However, the road to this transition is sprinkled with major technical unknowns such as long-term stability of the platform, reproducibility of the technology and clinical utility over traditional antibody-based platforms. Further, regulatory bodies that oversee the clinical laboratory operations are unfamiliar with this new technology. As a result, diagnostic laboratories have avoided using shotgun proteomics for routine diagnostics. In this perspectives article, we describe the clinical implementation of a shotgun proteomics assay for amyloid subtyping, with a special emphasis on standardizing the platform for better quality control and earning clinical acceptance. This assay is the first shotgun proteomics assay to receive regulatory approval for patient diagnosis. The blueprint of this assay can be utilized to develop novel proteomics assays for detecting numerous other disease pathologies.

Biochemical markers in early diagnosis and management of systemic amyloidoses

“…In particular, proteomic amyloid typing has already moved into clinical practice and has been implemented as a clinical test [75].…”

“…However, major differences among the methods exist, principally involving the strategies used to fractionate the tissue proteins prior to MS, and the data analysis criteria that allow classifying one of the identified proteins as the amyloid constituent. A widely employed approach is based on selection of amyloid-positive areas by laser capture dissection/microdissection (LCD/LCM), retrieval/extraction of proteins, and shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS analysis) [50,67,68,[75][76][77]. In the final step of the process, a pathologist calls the amyloid subtype from the list of identified proteins (which include both fibrillar and fibril-associated proteins), by considering the most abundant amyloidogenic species detected across all microdissections.…”

“…This problem is especially relevant in the case of immunoglobulin light chains and genetic protein variants, and may prevent identifying and assigning significant peptides belonging to the amyloidogenic species [76]. The use of composite protein databases, supplemented with known human immunoglobulin variant domains and known amyloidogenic mutations, can partially overcome this issue [75].…”

Mass spectrometry analysis for amyloidosis typing - is the future bright for its clinical implementation?

“…Furthermore, unlike immunohistochemistry, MS/MS identification is independent of protein conformation. With significant technical advantages over immunohistochemistry, MS has been proposed as the new gold standard for amyloid typing based on promising reports of diagnostic accuracy [6,7].…”

Advances in clinical applications of tissue proteomics: opportunities and challenges

“…Thus the stakes are high and accurate typing of amyloid deposits is critical for major management decisions. To address this important clinical problem, the author's group utilized a semi-quantitative bottom-up shotgun MS-based proteomics approach to develop clinical tests to diagnose and classify systemic and localized amyloidosis [10]. The first test was developed to address the cases where a diagnosis of amyloidosis was established on FFPE biopsy material but the etiology was unclear.…”