China
Africa
Explore chapters and articles related to this topic
Protein Engineering and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
Quantitative proteomics deal with relative quantitative measurement of protein expression between healthy versus diseased and treated versus control samples. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by mass spectrometry (MS) analysis, protein array-based, and MS signal intensity-based quantification methods are used for quantitative proteomics. Labeling of proteins by incorporating stable isotopes such as 15N or 14N nitrogen or stable isotope labeling with amino acids in culture (SILAC) such as 2H-Leucine, 13C-Lysine can be done to improve sensitivity of the protein detection. Interactome is the branch of proteomics dealing with the study of protein–protein interaction using methods such as protein affinity chromatography, immunoprecipitation, and phage display (Saraswathy and Ramalingam 2011).
Optimization of the linear quantification range of an online trypsin digestion coupled liquid chromatography–tandem mass spectrometry (LC–MS/MS) platform
Published in Instrumentation Science & Technology, 2018
Zsuzsanna Kuklenyik, Jeffrey I. Jones, Christopher A. Toth, Michael S. Gardner, James L. Pirkle, John R. Barr
For targeted quantitative measurements, complete digestion is not a priority, if purified proteins are available as calibration standards and stable isotope labeled cleavage peptide analogs are used as internal standards. For quantitative proteomics, reproducibility and linearity in the concentration range of interest are the main priorities. A way to achieve better reproducibility is using a flow-through immobilized enzyme reactor (IMER), or “plug flow” reactor, that is online coupled with a LC–MS/MS system.[101112131415] Precise control of digestion buffer flow rate and constant trypsin column void volume give integrated IMER-LC–MS/MS systems enhanced method reproducibility.[10111213] Furthermore, because of the short digestion time (typically 2–6 min) and immediate transfer for LC–MS/MS analysis, potential degradation of the peptides cleavage products and internal standard analogs are minimized without the need for sample reduction and alkylation.[10]
Omics to address the opportunities and challenges of nanotechnology in agriculture
Published in Critical Reviews in Environmental Science and Technology, 2021
Sanghamitra Majumdar, Arturo A. Keller
Two-dimensional gel electrophoresis (2DE) separates protein mixtures based on charge (isoelectric point) in the first dimension and by mass in the second dimension on 2D-gels, which in combination with MS have been used for decades for plant proteome profiling. However, it has its limitations owing to poor sensitivity, low reproducibility, and low throughput. Especially for plant whole lysates that contain an array of secondary metabolites like pigments and phenolics, these compounds can introduce streaking in a gel (Vannini et al., 2014). With rapid development in MS techniques, quantitative proteomics have evolved from gel-based to gel-free approaches. Like metabolomics, proteomics can also be categorized into untargeted and targeted method. Due to the exploratory nature of untargeted proteomics, it is the preferred approach to screen for candidate protein markers to elucidate plant responses to ENM exposure. This approach can facilitate high-throughput analysis of protein abundance across different ENM exposures, tissue types, stages of growth, physiological conditions and stress conditions (Hart-Smith et al., 2017). In a gel-based approach, proteins are separated by 2DE and the spots showing comparable differences are excised from the gel. The proteins in the gel fractions are digested into peptides, which are then characterized by LC-MS. A typical gel- and label-free quantitative proteomic analysis used for discovery studies employ a bottom-up approach, where the proteins in a sample are cleaved into peptides, which are then separated, identified and quantified using LC-MS/MS (Figure 3) (Hu et al., 2015). The steps and key challenges in proteomic analysis in ENM-plant interaction study are summarized below.