Two-Dimensional Difference Gel Electrophoresis

Blundon, Malachi A.; Ganesan, Vinitha; Redler, Brendan; Van, Phu T.; Minden, Jonathan S.

doi:10.1007/978-1-4939-8793-1_20

Cited by 20 publications

(11 citation statements)

References 32 publications

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“…Whole cell proteomic analysis was performed by using 2-D fluorescence difference gel electrophoresis (DIGE) recommended by The Ohio State University Proteomics Shared Resource [30]. U251 cells (1 × 10 7 ) were incubated with 20 mM NaCl or 20 mM LiCl for 24 h. Cell pellets were snap frozen and processed for DIGE analysis by The Ohio State University Mass Spectrometry and Proteomics Facility using the Ettan DIGE system.…”

Section: Methodsmentioning

confidence: 99%

Proteomic Analysis Implicates Vimentin in Glioblastoma Cell Migration

Nowicki

Hayes

Chiocca

et al. 2019

Cancers

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We previously showed lithium chloride (LiCl) and other inhibitors of glycogen synthase kinase-3 (GSK-3) including 6-bromo-indirubin-3-oxime (BIO), can block glioblastoma (GBM) cell migration. To investigate the mechanisms involved we used two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry to identify proteins altered after treatment of U251 GBM cells with 20 mM LiCl. Downregulation of the intermediate filament protein vimentin was the most significant change identified. Analysis of patient tumor samples revealed that vimentin is expressed abundantly in GBM, and is prognostic especially in lower grade tumors. Additionally, siRNA-mediated vimentin knockdown impaired GBM migration. Western blotting showed that treatment with LiCl or small molecule GSK-3 inhibitors led to the rapid downregulation of detergent soluble vimentin levels across a panel of GBM-derived cells. Fluorescence reactivation after photobleaching (FRAP) microscopy studies showed a significant reduction in the ability of the vimentin cytoskeleton to recover from photo-bleaching in the presence of LiCl or BIO. Biochemical studies revealed that GSK-3 and vimentin directly interact, and analysis of vimentin revealed a GSK-3 consensus phosphorylation site. We conclude that anti-migratory compounds with the ability to inhibit GSK-3 have effects on vimentin cytoskeletal dynamics, which may play a role in their anti-invasive activity.

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Section: Methodsmentioning

confidence: 99%

Proteomic Analysis Implicates Vimentin in Glioblastoma Cell Migration

Nowicki

Hayes

Chiocca

et al. 2019

Cancers

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“…The 2D-DIGE method was originally developed by Minden and co-workers [185,186,187] and has been frequently modified as an efficient protein biochemical tool for studying large-scale changes in protein expression and interaction patterns [182,188,189,190]. The application of the 2D-DIGE technique for the comparative analysis of the skeletal muscle proteome from human and animal specimens has been described in detail in various methods papers [191,192,193,194,195].…”

Section: Top-down Proteomics Of Contractile Proteins From Skeletalmentioning

confidence: 99%

Characterization of Contractile Proteins from Skeletal Muscle Using Gel-Based Top-Down Proteomics

et al. 2019

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The mass spectrometric analysis of skeletal muscle proteins has used both peptide-centric and protein-focused approaches. The term ‘top-down proteomics’ is often used in relation to studying purified proteoforms and their post-translational modifications. Two-dimensional gel electrophoresis, in combination with peptide generation for the identification and characterization of intact proteoforms being present in two-dimensional spots, plays a critical role in specific applications of top-down proteomics. A decisive bioanalytical advantage of gel-based and top-down approaches is the initial bioanalytical focus on intact proteins, which usually enables the swift identification and detailed characterisation of specific proteoforms. In this review, we describe the usage of two-dimensional gel electrophoretic top-down proteomics and related approaches for the systematic analysis of key components of the contractile apparatus, with a special focus on myosin heavy and light chains and their associated regulatory proteins. The detailed biochemical analysis of proteins belonging to the thick and thin skeletal muscle filaments has decisively improved our biochemical understanding of structure-function relationships within the contractile apparatus. Gel-based and top-down proteomics has clearly established a variety of slow and fast isoforms of myosin, troponin and tropomyosin as excellent markers of fibre type specification and dynamic muscle transition processes.

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“…The proposed method has similar sensitivity to electrophoretic and conventional LC-MS/MS methods but is superior in terms of simplicity and by way of identification of proteins. The specifications of each proteomics method ( Iwasaki et al, 2010 ; Zhang et al, 2013 ; Imai, 2014 ; Nakata et al, 2015 ; Blundon et al, 2019 ; Lee et al, 2019 ; Kobayashi et al, 2021 ) are summarized in Table 1 .…”

Section: Fluorogenic Derivatization-liquid Chromatography-tandem Mass Spectrometry Proteomics Methods (Fd-lc-ms/ms)mentioning

confidence: 99%

Recent Progress in FD-LC-MS/MS Proteomics Method

Kobayashi

Imai

2021

Front. Chem.

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Through the course of our bio-analytical chemistry studies, we developed a novel proteomics analysis method, FD-LC-MS/MS (fluorogenic derivatization-liquid chromatography-tandem mass spectrometry). This method consists of fluorogenic derivatization (FD), LC separation, and detection/quantification of the derivatized proteins, followed by isolation, tryptic digestion of the isolated proteins, and final identification of the isolated proteins using electrospray ionization nano-LC-MS/MS of the generated peptide mixtures with a probability-based protein identification algorithm. In this review, we will present various examples where this method has been used successfully to identify expressed proteins in individual human cells. FD-LC-MS/MS is also suitable for differential proteomics analysis. Here, two biological samples are treated by the same steps mentioned above, and the two chromatograms obtained are compared to identify peaks with different intensities (variation in protein levels). Associated peak fractions are then isolated, and the differentially expressed proteins between the two samples are identified by LC-MS/MS. Several biomarkers for cancers have been identified by FD-LC-MS/MS. For more efficient separation, nano-flow LC with a phenyl-bonded monolithic silica-based capillary column was adopted for cell-expressed intact protein analysis. The derivatized human cell proteins (K562) and yeast cell (Saccharomyces cerevisiae) proteins as model intact cell proteins were analyzed by nano-flow LC with fluorescence detection. More than 1,300 protein peaks were separated/detected from both cells. For straightforward comparison of multiple peak separation profiles, a novel type of chromatogram display, termed the “spiderweb” chromatogram, was developed. A nano-LC-FD-LC-mass spectrometry trial for molecular weight estimation of FD proteins has also been conducted.

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Two-Dimensional Difference Gel Electrophoresis

Cited by 20 publications

References 32 publications

Proteomic Analysis Implicates Vimentin in Glioblastoma Cell Migration

Proteomic Analysis Implicates Vimentin in Glioblastoma Cell Migration

Characterization of Contractile Proteins from Skeletal Muscle Using Gel-Based Top-Down Proteomics

Recent Progress in FD-LC-MS/MS Proteomics Method

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