Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM)

Canessa, Emily H.; Goswami, Mansi V.; Alayi, Tchilabalo Dilezitoko; Hoffman, Eric P.; Hathout, Yetrib

doi:10.1002/jms.4437

Cited by 12 publications

(26 citation statements)

References 27 publications

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“…To verify the high protein expression in M1 polarization of both human THP-1 and mouse RAW264.7 macrophages from the proteomics results, 16 of 43 up-regulated protein were arbitrarily selected for targeted quantitation by PRM. It has been proved that PRM is more accurate and reproducible compared with the relative quantification by western blot and immunofluorescence ( 41 ). Results showed all proteins were up-regulated in M1 cells, which was consistent with the global proteomics data ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

Comparative Proteomic Analysis of Polarized Human THP-1 and Mouse RAW264.7 Macrophages

Zou

et al. 2021

Front. Immunol.

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Macrophages can be polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2) in the immune system, performing pro-inflammatory and anti-inflammatory functions, respectively. Human THP-1 and mouse RAW264.7 cell line models have been widely used in various macrophage-associated studies, while the similarities and differences in protein expression profiles between the two macrophage models are still largely unclear. In this study, the protein expression profiles of M1 and M2 phenotypes from both THP-1 and RAW264.7 macrophages were systematically investigated using mass spectrometry-based proteomics. By quantitatively analyzing more than 5,000 proteins among different types of macrophages (M0, M1 and M2) from both cell lines, we identified a list of proteins that were uniquely up-regulated in each macrophage type and further confirmed 43 proteins that were commonly up-regulated in M1 macrophages of both cell lines. These results revealed considerable divergences of each polarization type between THP-1 and RAW264.7 macrophages. Moreover, the mRNA and protein expression of CMPK2, RSAD2, DDX58, and DHX58 were strongly up-regulated in M1 macrophages for both macrophage models. These data can serve as important resources for further studies of macrophage-associated diseases in experimental pathology using human and mouse cell line models.

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Section: Resultsmentioning

confidence: 99%

Comparative Proteomic Analysis of Polarized Human THP-1 and Mouse RAW264.7 Macrophages

Zou

et al. 2021

Front. Immunol.

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“…This will be vital for future clinical trials in DMD, allowing for more accurate benchmarking of results against well characterised BMD phenotypes. Analysis of immunostained muscle fibres for dystrophin is complementary to other techniques such as western blotting and mass spectrometry, providing different insights into the molecular efficacy of therapeutic intervention [1,12]. This is mainly due to the ability to assess dystrophin localisation at the sarcolemma of myofibres which is not achievable by either western blot or mass spectrometry.…”

Section: Discussionmentioning

confidence: 99%

A high–throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies

Scaglioni

Ellis

Catapano

et al. 2020

acta neuropathol commun

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The primary molecular endpoint for many Duchenne muscular dystrophy (DMD) clinical trials is the induction, or increase in production, of dystrophin protein in striated muscle. For accurate endpoint analysis, it is essential to have reliable, robust and objective quantification methodologies capable of detecting subtle changes in dystrophin expression. In this work, we present further development and optimisation of an automated, digital, highthroughput script for quantitative analysis of multiplexed immunofluorescent (IF) whole slide images (WSI) of dystrophin, dystrophin associated proteins (DAPs) and regenerating myofibres (fetal/developmental myosin-positive) in transverse sections of DMD, Becker muscular dystrophy (BMD) and control skeletal muscle biopsies. The script enables extensive automated assessment of myofibre morphometrics, protein quantification by fluorescence intensity and sarcolemmal circumference coverage, colocalisation data for dystrophin and DAPs and regeneration at the single myofibre and whole section level. Analysis revealed significant variation in dystrophin intensity, percentage coverage and amounts of DAPs between differing DMD and BMD samples. Accurate identification of dystrophin via a novel background subtraction method allowed differential assessment of DAP fluorescence intensity within dystrophin positive compared to dystrophin negative sarcolemma regions. This enabled surrogate quantification of molecular functionality of dystrophin in the assembly of the DAP complex. Overall, the digital script is capable of multiparametric and unbiased analysis of markers of myofibre regeneration and dystrophin in relation to key DAPs and enabled better characterisation of the heterogeneity in dystrophin expression patterns

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“…A feature of particular value with our approach is use of spiked-in standards to generate reference points common to all proteins within a study and ideally between studies. Use of reference peptides labelled in vivo , has proved particularly useful in this respect and spiking with fully labelled reference peptides offers the near-ideal prospect of expressing all values in molar terms raising the utopian vision of fully stoichiometric analysis of the inter-relationships of the component proteins [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy

Novak

Spathis

Dang

et al. 2021

JND

Self Cite

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Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystroglycan complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

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Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM)

Cited by 12 publications

References 27 publications

Comparative Proteomic Analysis of Polarized Human THP-1 and Mouse RAW264.7 Macrophages

Comparative Proteomic Analysis of Polarized Human THP-1 and Mouse RAW264.7 Macrophages

A high–throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy

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