Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

Chen, Hang; Hsiao, Yung-Chin; Chiang, Sum-Fu; Wu, Chia‐Chun; Lin, Yung Hsiang; Liu, Hsuan; Zhao, Hong; Chen, Jinn‐Shiun; Chang, Yu‐Sun; Yu, Jau‐Song

doi:10.1016/j.aca.2016.05.037

Cited by 7 publications

(5 citation statements)

References 43 publications

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“…Since the BRAF WT allele can continue to be expressed in BRAF V600E -mutant cancer cells (42–44), we reasoned that upstream activation of this pathway could recruit BRAF WT proteins to form dimers, thus engaging pathway components which possess relatively low kinase activity. To determine if BRAF WT :CRAF dimers could dampen BRAF V600E activity, we again used BRET assays to determine transfection conditions in HEK293T cells that yielded stimulation-dependent increases in V-BRAF WT :RLuc-CRAF heterodimers.…”

Section: Resultsmentioning

confidence: 99%

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling

et al. 2017

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The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF or NRAS activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a kit(lf) mutation into a strain of Tg(mitfa:BRAFV600E); p53(lf) melanoma-prone zebrafish. Melanoma onset was accelerated in kit(lf); Tg(mitfa:BRAFV600E); p53(lf) fish. Tumors from kit(lf) animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAFV600E-mutant human melanoma cell line. We found that pathway stimulation upstream of BRAFV600E could paradoxically reduce signaling downstream of BRAFV600E, and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAFV600E signaling. In vivo, expression of wild-type BRAF delayed melanoma onset, but only in a kit-dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAFV600E-driven melanoma formation.

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Section: Resultsmentioning

confidence: 99%

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling

et al. 2017

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“…In the course of this research we found only one mass spectrometry-based study that identified and quantified the mutated B-raf protein in tumor tissues [17]. The mutation was detected in two colorectal carcinoma tissues with 2 mg of total protein extract used as the starting material.…”

Section: Discussionmentioning

confidence: 99%

“…The identification and quantitation of B-raf V600E protein by mass spectrometry is challenging. To date, only one study has attempted to address the issues of heterogeneity that is inherent to expression of WT and V600E BRAF [17]. Using immunoenrichment-based techniques following a targeted liquid chromatography multiple reaction monitoring (LC-MRM) approach, both proteins were identified and quantitated in complex biological samples comprised of colorectal carcinoma CRC cell lines and tissue specimens.…”

Section: Introductionmentioning

confidence: 99%

The Hidden Story on Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma – Association with Clinical Outcome and Tumor Phenotypes

Betancourt¹,

Szász²,

Kuras³

et al. 2019

Preprint

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In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas do clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression for the mutated protein is associated with a more aggressive tumor progression. Our study design that is comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis may enable the eventual delineation of patient responders/non-responders and the subsequent therapy of malignant melanoma.

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“…Targeted proteomics has also been used for quantification of mutant proteins in other diseases, such as GLUT1 P485L , ITPR1 P1059L , and CACNA1H P648L for dileucineopathies (Meyer et al, 2018), TEX101 G99V in infertility (Schiza et al, 2019), and KRAS G13D and BRAF V600E in colon cancer (Chen et al, 2016; Demory Beckler et al, 2013; Lin et al, 2019). With gel electrophoresis fractionation, SRM‐based targeted proteomics was applied for detection and quantification of SP‐A variants (Gln223 and Lys223) from bronchoalveolar lavage (BAL) (Foster et al, 2014), GFAP variants (R79C, R239H, and R416W) for Alexander disease (Heaven et al, 2019), and CYP21A2 variants (L388R, E140K, P45L, V211M, and V281L) from congenital adrenal hyperplasia (CAH) patient plasma (Brønstad et al, 2014).…”

Section: Targeted Proteomics For Analysis Of Protein Mutationsmentioning

confidence: 99%

Mass spectrometry‐based targeted proteomics for analysis of protein mutations

Lin

Zhang

Kitata

et al. 2021

Mass Spectrometry Reviews

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Cancers are caused by accumulated DNA mutations. This recognition of the central role of mutations in cancer and recent advances in next‐generation sequencing, has initiated the massive screening of clinical samples and the identification of 1000s of cancer‐associated gene mutations. However, proteomic analysis of the expressed mutation products lags far behind genomic (transcriptomic) analysis. With comprehensive global proteomics analysis, only a small percentage of single nucleotide variants detected by DNA and RNA sequencing have been observed as single amino acid variants due to current technical limitations. Proteomic analysis of mutations is important with the potential to advance cancer biomarker development and the discovery of new therapeutic targets for more effective disease treatment. Targeted proteomics using selected reaction monitoring (also known as multiple reaction monitoring) and parallel reaction monitoring, has emerged as a powerful tool with significant advantages over global proteomics for analysis of protein mutations in terms of detection sensitivity, quantitation accuracy and overall practicality (e.g., reliable identification and the scale of quantification). Herein we review recent advances in the targeted proteomics technology for enhancing detection sensitivity and multiplexing capability and highlight its broad biomedical applications for analysis of protein mutations in human bodily fluids, tissues, and cell lines. Furthermore, we review recent applications of top‐down proteomics for analysis of protein mutations. Unlike the commonly used bottom‐up proteomics which requires digestion of proteins into peptides, top‐down proteomics directly analyzes intact proteins for more precise characterization of mutation isoforms. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large‐scale targeted detection and quantification of important protein mutations are discussed.

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Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

Cited by 7 publications

References 43 publications

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling

The Hidden Story on Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma – Association with Clinical Outcome and Tumor Phenotypes

Mass spectrometry‐based targeted proteomics for analysis of protein mutations

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Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

Cited by 7 publications

References 43 publications

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling

The Hidden Story on Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma &ndash; Association with Clinical Outcome and Tumor Phenotypes

Mass spectrometry‐based targeted proteomics for analysis of protein mutations

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The Hidden Story on Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma – Association with Clinical Outcome and Tumor Phenotypes