A Pilot Study of Rare Renal Amyloidosis Based on FFPE Proteomics.
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ABSTRACT:
SUBMITTER: Meng S
PROVIDER: S-EPMC8659071 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Amyloidosis is a rare disease caused by the misfolding and extracellular aggregation of proteins as insoluble fibrillary deposits localized either in specific organs or systemically throughout the body. The organ targeted and the disease progression and outcome is highly dependent on the specific fibril-forming protein, and its accurate identification is essential to the choice of treatment. Mass spectrometry-based proteomics has become the method of choice for the identification of the amyloidogenic protein. Regrettably, this identification relies on manual and subjective interpretation of mass spectrometry data by an expert, which is undesirable and may bias diagnosis. To circumvent this, we developed a statistical model-assisted method for the unbiased identification of amyloid-containing biopsies and amyloidosis subtyping. Based on data from mass spectrometric analysis of amyloid-containing biopsies and corresponding controls. A Boruta method applied on a random forest classifier was applied to proteomics data obtained from the mass spectrometric analysis of 75 laser dissected Congo Red positive amyloid-containing biopsies and 78 Congo Red negative biopsies to identify novel "amyloid signature" proteins that included clusterin, fibulin-1, vitronectin complement component C9 and also three collagen proteins, as well as the well-known amyloid signature proteins apolipoprotein E, apolipoprotein A4, and serum amyloid P. A SVM learning algorithm were trained on the mass spectrometry data from the analysis of the 75 amyloid-containing biopsies and 78 amyloid-negative control biopsies. The trained algorithm performed superior in the discrimination of amyloid-containing biopsies from controls, with an accuracy of 1.0 when applied to a blinded mass spectrometry validation data set of 103 prospectively collected amyloid-containing biopsies. Moreover, our method successfully classified amyloidosis patients according to the subtype in 102 out of 103 blinded cases. Collectively, our model-assisted approach identified novel amyloid-associated proteins and demonstrated the use of mass spectrometry-based data in clinical diagnostics of disease by the unbiased and reliable model-assisted classification of amyloid deposits and of the specific amyloid subtype.
| S-EPMC8745254 | biostudies-literature
Project description:Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.
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Project description:BackgroundEarly diagnosis and typing are crucial for improving the prognosis of patients with renal amyloidosis. Currently, Untargeted proteomics based precise diagnosis and typing of amyloid deposits are crucial for guiding patient management. Although untargeted proteomics achieve ultra-high-throughput by selecting the most abundant eluting cationic peptide precursors in series for tandem MS events, it lacks in sensitivity and reproducibility, which may not be suitable for early-stage renal amyloidosis with minor damages. Here, we aimed to develop parallel reaction monitoring (PRM)-based targeted proteomics to achieve high sensitivity and specificity by determining absolute abundances and codetecting all transitions of highly repeatable peptides of preselected amyloid signature and typing proteins in identifying early-stage renal immunoglobulin-derived amyloidosis.Methods and resultsIn 10 discovery cohort cases, Congo red-stained FFPE slices were micro-dissected and analyzed by data-dependent acquisition-based untargeted proteomics for preselection of typing specific proteins and peptides. Further, a list of proteolytic peptides from amyloidogenic proteins and internal standard proteins were quantified by PRM-based targeted proteomics to validate performance for diagnosis and typing in 26 validation cohort cases. The diagnosis and typing effectiveness of PRM-based targeted proteomics in 10 early-stage renal amyloid cases was assessed via a comparison with untargeted proteomics. A peptide panel of amyloid signature proteins, immunoglobulin light chain and heave chain in PRM-based targeted proteomics showed significantly distinguishing ability and amyloid typing performance in patients. The diagnostic algorithm of targeted proteomics with a low amount of amyloid deposits in early-stage renal immunoglobulin-derived amyloidosis showed better performance than untargeted proteomics in amyloidosis typing.ConclusionsThis study demonstrates that the utility of these prioritized peptides in PRM-based targeted proteomics ensure high sensitivity and reliability for identifying early-stage renal amyloidosis. Owing to the development and clinical application of this method, rapid acceleration of the early diagnosis, and typing of renal amyloidosis is expected.
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Project description:Introduction: Systemic amyloidosis is a diverse group of diseases that, although rare, pose a serious health issue and can lead to organ failure and death. Amyloid typing is essential in determining the causative protein and initiating proper treatment. Mass spectrometry-based proteomics is currently the most sensitive and accurate means of typing amyloid. Areas covered: Amyloidosis can be systemic or localized, acquired or hereditary, and can affect any organ or tissue. Diagnosis requires biopsy, histological analysis, and typing of the causative protein to determine treatment. The kidneys are the most commonly affected organ in systemic disease. Fibrinogen alpha chain amyloidosis (AFib) is the most prevalent form of hereditary renal amyloidosis. Select mutations in the fibrinogen Aα (FGA) gene lead to AFib. Expert commentary: Mass spectrometry is currently the most specific and sensitive method for amyloid typing. Identification of the mutated fibrinogen alpha chain can be difficult in the case of 'private' frameshift mutations, which dramatically change the sequences of the expressed fibrinogen alpha chain. A combination of expert pathologist review, mass spectrometry, and gene sequencing can allow for confident diagnosis and determination of the fibrinogen alpha chain mutated sequence.
| S-EPMC6788741 | biostudies-literature
Project description:Chronic alcohol exposure can cause myocardial degenerative diseases, manifested as cardiac insufficiency, arrhythmia, etc. These are defined as alcoholic cardiomyopathy (ACM). Alcohol-mediated myocardial injury has previously been studied through metabolomics, and it has been proved to be involved in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway concerning unsaturated fatty acids biosynthesis and oxidative phosphorylation, which tentatively explored the mechanism of ACM induced by chronic drinking. To further study alcohol-induced myocardial injury, myocardial specimens from a previously successfully established mouse model of ACM were subjected to histological, echocardiographic, and proteomic analyses, and validated by real-time quantitative polymerase chain reaction (qPCR). Results of histopathology and echocardiography showed the hypertrophy of cardiomyocytes, the dilation of ventricles, and decreased cardiac function. Proteomic results, available via ProteomeXchange with identifier PXD032949, revealed 56 differentially expressed proteins (DEPs) were identified, which have the potential to be involved in the KEGG pathway related to fatty acid biosynthesis disorders, lipid metabolism disorders, oxidative stress, and, ultimately, in the development of dilated cardiomyopathy (DCM). The present study further elucidates the underlying effects of myocardial injury due to chronic alcohol intake, laying a foundation for further studies to clarify the potential mechanisms of ACM.
| S-EPMC9268295 | biostudies-literature
Project description:Primary cutaneous amyloidosis (PCA) is a localized skin disorder that is characterized by the abnormal deposition of amyloid in the extracellular matrix (ECM) of the dermis. The pathogenesis of PCA is poorly understood. The objective of the present study was to survey proteome changes in PCA lesions in order to gain insight into the molecular basis and pathogenesis of PCA. Total protein from PCA lesions and normal skin tissue samples were extracted and analyzed using the isobaric tags for relative and absolute quantitation technique. The function of differentially expressed proteins in PCA were analyzed by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction analysis. The proteins that were most upregulated in PCA lesions were further analyzed by immunohistochemistry. A total of 1,032 proteins were identified in PCA lesions and control skin samples, with 51 proteins differentially expressed in PCA lesions, of which 27 were upregulated. In PCA lesions, the upregulated proteins were primarily extracellulary located. In addition, GO analysis indicated that the upregulated proteins were significantly enriched in the biological processes of epidermal development, collagen fiber organization and response to wounding (adjusted P<0.001). KEGG analysis indicated that the upregulated proteins were significantly enriched in the signaling pathways of cell communication, ECM receptor interaction and focal adhesion (adjusted P<0.001). Furthermore, the upregulated proteins were enriched in the molecular function of calcium ion binding, and the calcium binding proteins calmodulin-like protein 5, S100 calcium-binding protein A7 (S100A7)/fatty-acid binding protein and S100A8/A9 exhibited the highest levels of upregulation in PCA. This analysis of differentially expressed proteins in PCA suggests that increased focal adhesion, differentiation and wound healing is associated with the pathogenesis of PCA.
| S-EPMC5585729 | biostudies-literature
Project description:Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing an invaluable resource for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present high-yield protein extraction and recovery by direct solubilization (HYPERsol), an optimized workflow using ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936). When applied to archival samples, HYPERsol resulted in high-quality data from FFPE specimens in storage for up to 17 years, and may enable the discovery of new immunohistochemical markers.
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Project description:BackgroundCerebral amyloid angiopathy (CAA) occurs in 80% of patients with Alzheimer's disease (AD) and is mainly caused by the abnormal deposition of Aβ in the walls of cerebral blood vessels. Cerebrovascular molecular mechanisms in CAA were investigated by using comprehensive and accurate quantitative proteomics.MethodsConcerning the molecular mechanisms specific to CAA, formalin-fixed paraffin-embedded (FFPE) sections were prepared from patients having AD neuropathologic change (ADNC) with severe cortical Aβ vascular deposition (ADNC +/CAA +), and from patients having ADNC without vascular deposition of Aβ (ADNC +/CAA -; so called, AD). Cerebral cortical vessels were isolated from FFPE sections using laser microdissection (LMD), processed by pressure cycling technology (PCT), and applied to SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics.ResultsThe protein expression levels of 17 proteins in ADNC +/CAA +/H donors (ADNC +/CAA + donors with highly abundant Aβ in capillaries) were significantly different from those in ADNC +/CAA - and ADNC -/CAA - donors. Furthermore, we identified 56 proteins showing more than a 1.5-fold difference in average expression levels between ADNC +/CAA + and ADNC -/CAA - donors, and were significantly correlated with the levels of Aβ or Collagen alpha-2(VI) chain (COL6A2) (CAA markers) in 11 donors (6 ADNC +/CAA + and 5 ADNC -/CAA -). Over 70% of the 56 proteins showed ADNC +/CAA + specific changes in protein expression. The comparative analysis with brain parenchyma showed that more than 90% of the 56 proteins were vascular-specific pathological changes. A literature-based pathway analysis showed that 42 proteins are associated with fibrosis, oxidative stress and apoptosis. This included the increased expression of Heat shock protein HSP 90-alpha, CD44 antigen and Carbonic anhydrase 1 which are inhibited by potential drugs against CAA.ConclusionsThe combination of LMD-based isolation of vessels from FFPE sections, PCT-assisted sample processing and SWATH analysis (FFPE-LMD-PCT-SWATH method) revealed for the first time the changes in the expression of many proteins that are involved in fibrosis, ROS production and cell death in ADNC +/CAA + (CAA patients) vessels. The findings reported herein would be useful for developing a better understanding of the pathology of CAA and for promoting the discovery and development of drugs and biomarkers for CAA.
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Project description:Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.
| S-EPMC7554798 | biostudies-literature
Project description:Background and Objective: Mild hypospadias is a birth congenital condition characterized by the relocation of the male urethral meatus from its typical anatomical position near the tip of the glans penis, to a lower ventral position up to the brim of the glans corona, which can also be accompanied by foreskin ventral deficiency. For the most part, a limited number of cases have known etiology. We have followed a high-throughput proteomics approach to study the proteome in mild hypospadias patients. Methods: Foreskin samples from patients with mild hypospadias were collected during urethroplasty, while control samples were collected during elective circumcision (n = 5/group). A high-throughput, quantitative proteomics approach based on multiplexed peptide stable isotope labeling (SIL) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to ascertain protein abundance changes in hypospadias patients when compared to control samples. Results: A total of 4,815 proteins were quantitated (2,522 with at least two unique peptides). One hundred and thirty-three proteins from patients with mild hypospadias showed significant abundance changes with respect to control samples, where 38 proteins were increased, and 95 proteins were decreased. Unbiased functional biological analysis revealed that both mitochondrial energy production and apoptotic signaling pathways were enriched in mild hypospadias. Conclusions: This first comprehensive proteomics characterization of mild hypospadias shows molecular changes associated with essential cellular processes related to energy production and apoptosis. Further evaluation of the proteome may expand the search of novel candidates in the etiology of mild hypospadias and could also lead to the identification of biomarkers for this congenital urogenital condition.
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