China
Africa
Explore chapters and articles related to this topic
The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
The window of separation and identification using these techniques is limited by the size of the proteins, their pH, and their abundance. Also, the overall approach is labor-intensive, time-consuming and difficult to automate, and can suffer from poor reproducibility (e.g., variability in the enzymatic digestions, ion interference, lack of standards, gel-to-gel variations, etc.) which adds to the complexity. A related methodology known as Fluorescence 2D Difference Gel Electrophoresis (fluorescence 2D DIGE) can overcome some of the problems by using unique fluorescent dyes (e.g., Cy2, Cy3, Cy5) to label the proteins of different samples which can be run on the same gel and quantified simultaneously. Thus, the throughput can be doubled or tripled compared to the classical 2D PAGE method, and gel-to-gel variations can be smoothed.
Pharmacogenomics of Colorectal Cancer
Published in Jim Cassidy, Patrick Johnston, Eric Van Cutsem, Colorectal Cancer, 2006
Patrick Johnston, Howard L. McLeod
A study by Friedman et al. made use of 2D difference gel electrophoresis coupled with mass spectrometry to investigate tumor-specific changes in the proteosome of human colorectal cancer and normal mucosa. The study was extremely small, as it contained only six samples, but the improved method has the potential to facilitate future proteomic profiling of large sets of colorectal cancer samples. In this method, the tumor samples and normal samples are labeled with cy3 and cy5 fluorescent dyes. The method allows each patient comparison to be preformed on proteins resolved in the same 2D gel separation, thereby removing the error caused by gel–gel variation and allows quantification of abundance of change for each protein pair (153).
Proteomics Approaches to Uncover the Drug Resistance Mechanisms of Microbial Biofilms
Published in Chaminda Jayampath Seneviratne, Microbial Biofilms, 2017
Chaminda Jayampath Seneviratne, Tanujaa Suriyanarayanan, Lin Qingsong, Juan Antonio Vizcaíno
Significant advances in gel-based proteomics were made in the 1990s with the introduction of the 2D-difference gel electrophoresis (2D-DIGE) technique by Unlu’s research group. In 2D-DIGE, complex protein mixtures are labelled with fluorescent dyes (e.g. Cy2, Cy4 and Cy5), before 2-DE [100,101]. There are two major advantages of 2D-DIGE over traditional 2-DE methods. First, as many as three samples can be run in a single gel, thereby reducing experimental variations and making protein quantification more accurate. Second, gel-to-gel variations can be resolved by introducing an internal control. DIGE has been successfully applied in both bacterial and fungal biofilm studies (Figure 6.1b) [20,40,102,103]. However, gel-based approaches are limited by several technical hurdles, such as in the case of resolving hydrophobic proteins and low-abundance proteins. In particular, the first-dimension isoelectric focusing in conventional 2-DE platforms is not compatible enough with hydrophobic proteins and proteins with extreme isoelectric points. The second-dimension and/or the different staining techniques are not sufficiently sensitive to recover low molecular weight and low-abundance proteins. Other drawbacks are the limited detection of membrane proteins caused by their poor solubility and the limited dynamic range. Overall, lack of reproducibility, co-migration of proteins in spots and limited dynamic range are some of the major problems in gel-based proteomics [104,105]. In addition, these methods are labor-intensive, time-consuming and difficult to automate. Therefore, as previously mentioned, most researchers have already moved to gel-free platforms for proteomics analysis.
Proteomic approaches to assist in diagnosis and prognosis of oral cancer
Published in Expert Review of Proteomics, 2021
Jamile De Oliveira Sá, Luciana Daniele Trino, Ana Karina Oliveira, Ariane Fidelis Busso Lopes, Daniela Campos Granato, Ana Gabriela Costa Normando, Erison Santana Santos, Leandro Xavier Neves, Carolina Moretto Carnielli, Adriana Franco Paes Leme
The application of MS-based proteomics in the study of OSCC provided evidence that the proteolytic webs are extensively modulated in the context of oral cancer. Commonly applied in the early 2000s, two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel digestion and MS analysis allowed for the first attempts toward global proteome characterization in a different biological context. In OSCC, a 2D-DIGE analysis of HSC-2 and HSC-3 cell lines on human normal oral keratinocytes revealed 22 differential proteins with ≥2-fold change. Among 13 proteins with reduced abundance in OSCC cell lines, the SERPINB5 was identified using MALDI-TOF [90]. Serpin B3 was also found increased in tumor tissues compared to surrounding mucosa using 2D-DIGE-MALDI-TOF [91], or OSCC tissues compared to control [92], or in the serum of orthotopic mouse model inoculated with Tca8113 cell line and in the serum of OSCC patients [93]. 2D-DIGE followed by ESI-LC-MS/MS revealed that Serpin B1 cellular levels correlate with pro-migratory phenotype in OSCC cell lines [94], while SELDI-TOF ProteinChip platform analysis pointed up the cysteine endopeptidase inhibitor cystatin SA-I as a potential saliva biomarker of OSCC [95].
Utilizing proteomics to understand and define hypertension: where are we and where do we go?
Published in Expert Review of Proteomics, 2018
Christian Delles, Emma Carrick, Delyth Graham, Stuart A. Nicklin
We cannot provide a comprehensive overview of proteomic techniques in this review but would like to briefly put experimental and clinical data into the context of contemporary proteomic techniques. Readers are referred to review articles providing further background, including a recent American Heart Association statement [18]. The term proteomics was first coined in 1994 by Marc Wilkins as the study of an organism’s proteome at a given time [19]. Techniques involved in proteomics include, but are not limited to, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2D-difference gel electrophoresis (2D-DIGE), immunoassays, protein microarrays, and variations of mass spectrometry. 2D gel electrophoresis was used to observe that ferritin light chain was significantly increased in coronary artery disease [20]. 2D protein gel databases for human and other animal models have been created and used for reference [21].
Advances in the proteomics of amniotic fluid to detect biomarkers for chromosomal abnormalities and fetomaternal complications during pregnancy
Published in Expert Review of Proteomics, 2019
Aayushi Vasani, Maushmi S. Kumar
One-dimensional (1D) and two-dimensional (2D) sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) has been commonly used for separation of protein mixtures in combination with chromatographic separations. Two-dimensional gel electrophoresis (2DGE) preceded and led to the birth of current age proteomics. In 2DGE, proteins are separated based on two principles-isoelectric pH and molecular weight followed by digestion, which is performed with the help of enzymes like trypsin, chymotrypsin, etc. It is also used to study post-translational modification (PTM) represented by phosphorylation, glycosylation, glutathionylation, etc. [7,8]. However, 2DGE has some disadvantages, as it can be time-consuming and is less efficient in separating less abundant proteins. To overcome these shortcomings, a new classic technique named as DIGE (Difference gel electrophoresis) is introduced; this form of gel electrophoresis uses up to three different protein samplers labeled with size-matched spectrally resolvable fluorescent dyes (Cy3, Cy5, Cy2) prior to 2DGE. This reduces the variability factor by labeling different samples with different dyes followed by image analysis with 2D platinum [8]. This technique, if used along with MS or MALDI-TOF, increases the extent of protein identification and quantitates even minuscule difference between the samples induced by any biological events. After separation of protein mixture in 2DGE, fragmentation and the identification of ionized products is performed using mass spectrometry (MS), wherein charged particles or ions are separated according to their mass/charge (m/z) ratio.