Proteomic Analysis for the Diagnosis of Fibrinogen Aα-chain Amyloidosis

Abstract

Introduction: Hereditary fibrinogen Aa-chain (AFib) amyloidosis is a relatively uncommon renal disease associated with a small number of pathogenic fibrinogen Aa (FibA) variants; wild-type FibA normally does not result in amyloid deposition. Proteomics is now routinely used to identify the amyloid type in clinical samples, and we report here our algorithm for identification of FibA in amyloid. Methods: Proteomics data from 1001 Congo red-positive patient samples were examined using the Mascot search engine to interrogate the Swiss-Prot database and generate protein identity scores. An algorithm was applied to identify FibA as the amyloid protein based on Mascot scores. FibA variants were identified by appending the known amyloidogenic variant sequences to the Swiss-Prot database. Results: AFib amyloid was identified by proteomics in 64 renal samples based on the Mascot scores relative to other amyloid proteins, the presence of a pathogenic variant, and coverage of the p.449-621 sequence. Contamination by blood could be excluded from a comparison of the FibA score with that of the fibrinogen b and g chains. The proteomics results were consistent with the clinical diagnosis. Four additional renal samples did not fulfill all the criteria using the algorithm but were adjudged as AFib amyloid based on a full assessment of the clinical and biochemical results. Conclusion: AFib amyloid can be identified reliably in glomerular amyloid by proteomics using a scorebased algorithm. Proteomics data should be used as a guide to AFib diagnosis, with the results considered together with all available clinical and laboratory information.

Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre

“…In some cases, this may be a simple misidentification based on a low scoring variant peptide, however, the possibility of mosaicism cannot be ruled out, with a low level acquired clone generating low plasma concentrations of a highly amyloidogenic variant protein which is preferentially deposited in amyloid. Similar issues were observed in a study of renal fibrinogen Aα amyloid [48]; here we observed 3 cases where two variants were observed by proteomics although only one was identified by gene sequencing. In the example shown in Figure 2C Another area where diagnostic amyloid proteomics could be improved is in the identification of immunoglobulin light and heavy chain variant regions.…”

“…Here, trypsin digestion results in the release of both amyloid and thrombusderived peptides, and this may confound amyloid fibril protein identification. In the case fibrinogen Aα (FibA) amyloid, we can differentiate the amyloid protein from contaminating blood by including the scores of fibrinogen B and G chains and the presence of an amyloidogenic variant into our algorithm [48]. In other cases, a range of proteins can be Tissue decellularisation is one approach to remove such ambiguities.…”

“…In principle, structural information is already available from the MS data and can be extracted by appending a list of variants to the Swiss-Prot database before searching. Proteomics for variant identification has been invaluable in some cases, particularly in the correct identification of fibrinogen Aα amyloid [48] where we have added an extra set of 12 variants, including the C terminal "VLITLG" base deletion variants [57,58] There are also a number of factors which could lead to misidentification of a variant, the most obvious being where the search engine conjoins peptides from the wild-type protein with a misidentified peptide derived from another protein. A high probability score for a protein does not necessarily equate with a high probability identity for the variant.…”

A Stepwise Data Interpretation Process For Renal Amyloidosis Subtyping by LMD-MS

“…The prevalence rate of different amyloidosis subtypes vary greatly in different regions, but anyway, AL is always the most popular one (59%-68%); serum amyloid A amyloidosis (AA, 4-12%), ALECT2 (~3%) and transthyretin amyloidosis (ATTR, 3%-33%) could be considered as sub-popular subtypes; whereas the other subtypes are rare [1,23,24]. In AL, κ-type (ALκ) and λ-type (ALλ) interfere with each other, mainly because of the high background of κ chain; In terms of subtyping brinogen α chain amyloidosis (AFib), the SC of FIBA should be greater than the sum of brinogen β chain (FIBB) and brinogen γ chain (FIBG) in addition to the requirement of the highest SC of FIBA among all amyloid proteins [25]; and as mentiioned above, ALECT2 is a special existence. Besides, special considerations are required for making de nite diagnosis of immunoglobulin heavy chain amyloidosis (AH) and its involvement with AL, that is immunoglobulin heavy-light chain amyloidosis (AHL) [26].…”

Lysozyme amyloid: evidence for the W64R variant by proteomics in the absence of the wild type protein

“…A sample of Congo red positive formalin-fixed paraffin-embedded (FFPE) GI tissue was collected by laser capture dissection and analysed by proteomics using trypsin as the digestion enzyme [ 5 ]. Data were then evaluated using MASCOT software to search the Swiss-Prot database to which was appended an additional lysozyme database of nine variants.…”