Dataset Information

Subtyping of cardiac amyloidosis by mass spectrometry-based proteomics of endomyocardial biopsies

ABSTRACT: Cardiac amyloidosis is a severe clinical condition leading to restrictive cardiomyopathy and heart failure. With increasing options for specific treatment of different cardiac amyloid diseases, correct amyloid subtyping is essential for clinical outcome. Mass spectrometry-based methods for cardiac amyloid subtyping have become important diagnostic tools but are currently used only in a few reference laboratories. Such methods typically include laser-capture microdissection to ensure the specific analysis of amyloid deposits. While this strategy effectively reduces background signals, it is a labor-intensive and costly procedure hampering the implementation of these methods into a wider group of laboratories. Here we introduce a more direct proteomics-based method for subtyping of cardiac amyloids. Endomyocardial biopsies were retrospectively analyzed from fresh frozen material of 78 patients with cardiac amyloidosis and from 12 biopsies of unused donor heart explants. Cryostat sections were directly digested with trypsin and analyzed with the nano-liquid chromatography (nLC-MS/MS), and the data were evaluated by a proteomic software. With a diagnostic threshold set to 70% for each of the four most common amyloid proteins affecting the heart (LC k, LC l, TTR and SAA), 65 of the cases (87%) could be diagnosed, and of these, 61 cases (94%) were in concordance with the original diagnoses. The specimens were also analyzed for the summed intensities of the amyloid signature proteins (ApoE, ApoA-IV and SAP). The summed intensities were significantly higher (p<0.001) for all assigned amyloid cases, whereas unassigned cases were not significantly different from controls and had lower signals than the assigned cases (p<0.001), suggesting that signature proteins can serve as in situ quality markers of cardiac amyloid deposits. We argue that this efficient workflow can be useful for disseminating proteomics for cardiac amyloid subtyping into accredited clinical laboratories.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Heart

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Proteomics Core Facility

LAB HEAD: Carina Sihlbom

PROVIDER: PXD033168 | Pride | 2022-10-27

REPOSITORIES: Pride

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Publications

Subtyping of cardiac amyloidosis by mass spectrometry-based proteomics of endomyocardial biopsies.

Noborn Fredrik F Thomsen Christer C Vorontsov Egor E Bobbio Emanuele E Sihlbom Carina C Nilsson Jonas J Polte Christian L CL Bollano Entela E Vukusic Kristina K Sandstedt Joakim J Dellgren Göran G Karason Kristjan K Oldfors Anders A Larson Göran G

Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis 20221009 1

<h4>Background</h4>Cardiac amyloidosis is a severe condition leading to restrictive cardiomyopathy and heart failure. Mass spectrometry-based methods for cardiac amyloid subtyping have become important diagnostic tools but are currently used only in a few reference laboratories. Such methods include laser-capture microdissection to ensure the specific analysis of amyloid deposits. Here we introduce a direct proteomics-based method for subtyping of cardiac amyloidosis.<h4>Methods</h4>Endomyocardi ...[more]

PMID: 36209425

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Project description:This track is produced as part of the ENCODE Project. This track shows genome-wide assessment of DNA replication timing in cell lines using the sequencing-based "Repli-seq" methodology (see below). Replication timing is known to be an important feature for epigenetic control of gene expression that usually operates at a higher-order level than at the level of specific genes. For each experiment (cell line, replicate), replication timing was ascertained by the isolation and sequencing of newly replicated DNA from six cell cycle fractions: G1/G1b, S1, S2, S3, S4, G2 (six fraction profile). Replication patterns are visualized as a continuous function based on sequencing tag density (Percentage-normalized Signal) and as a wavelet-smoothed transform of the six fraction profile (Wavelet-smoothed Signal). Replication peaks corresponding to replication initiation zones (Peaks) and valleys corresponding to replication termination zones (Valleys) were determined from local maxima and minima, respectively, in the wavelet-smoothed signal data. A measure of relative copy number at each genomic location (Summed Densities) was determined by summing the normalized tag density values of each cell cycle fraction at that location (equals one replicated genome equivalent). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. Repli-seq was performed as described by Hansen et al. (2010). Briefly, newly replicated DNA was labeled in vivo with a pulse of 5-bromo-2-deoxyuridine (BrdU), cells were fractionated into six different parts of the cell cycle by flow cytometry according to DNA content, cell cycle fractionated DNA was sonicated and an anti-BrdU monoclonal antibody was used to isolate the newly replicating DNA. Fragment ends were sequenced using the Illumina Genome Analyzer II or HiSeq platforms (36 bp reads). Some experiments (BJ, K562, BG02ES, GM06990) were originally performed and mapped to an earlier version of the human reference genome NCBI36/hg18 (Hansen et al., 2010) and were remapped to the more recent reference genome GRCh37/hg19. Uniquely mapping high-quality reads were mapped to the genome minus the Y chromosome. Replication signals within each six cell cycle fraction were derived from the density of sequence tags mapping within a 50 kb sliding window (stepped 1 kb across the genome); these densities were normalized to 4 million tags per genome. To avoid variation due to copy number or sequence bias, cell cycle-specific replication signals at each location were determined as a percentage of the sum of the six normalized tag density signals (Percentage-normalized Signal). To transform the six fraction replication signals into one track (Wavelet-smoothed Signal), the percentage-normalized signals at each location were used to calculate a weighted average value based on the average DNA content of each fraction according to flow cytometry [higher values correspond to earlier replication; formula=(0.917*G1b)+(0.750*S1)+(0.583*S2)+(0.417*S3)+(0.250*S4)+(0*G2)]. These weighted average data were smoothed by wavelet transformation [J7 level, corresponding to a scale of 128 kb; see Thurman et al. (2007)]. Replication initiation zones were flagged by determining local maxima in the wavelet-smoothed data (Peaks) and, similarly, replication termination zones were flagged by local minima (Valleys). The sum of the 4 million normalized replication tag densities correspond to replication of one genome and can, therefore, be used as a measure of relative genomic copy number (Summed Densities). This is useful for evaluation of unusual replication patterns, such as "biphasic" ones where replication has both early and late components [as described by Hansen et al. (2010)].

Dataset Information

Subtyping of cardiac amyloidosis by mass spectrometry-based proteomics of endomyocardial biopsies

Publications

Subtyping of cardiac amyloidosis by mass spectrometry-based proteomics of endomyocardial biopsies.

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