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Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
quantitated using PDQuest™ software. Spots indicating differentially expressed proteins were excised from the gel, destained, washed and subjected to in-gel digestion by trypsin. The extracted peptides were desalted by micro-reversed phase chromatography (Zip-Tip™) and analyzed by MALDI-TOF MS using a Bruker IVTM (Bruker Daltonics) in positive ion mode. The spectra were analyzed using MoverZ™ or MassLynx™, and peptide mass fingerprinting (PMF) was performed using MASCOT against the NCBInr database. For Western blotting, proteins were transferred to a PVDF membrane (Millipore) and probed with polyclonal rabbit anti-human immunoglobulin light chain or rabbit anti-human transthyretin antibodies (Dako). To obtain the amino acid sequence of the monoclonal light chains, cDNA was synthesized from total RNA in bone marrow plasma cells, amplified and sequenced. Mutations in TTR were detected by complete gene sequencing.
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
In a recent study, the biofilm formation determinants of Enterococcus faecalis were characterised using a traditional proteomics approach [38]. Interestingly, they selected one biofilm-efficient strain, which formed stronger and denser biofilm and one biofilm-deficient strain, which formed meagre biofilm, and examined them together with Enterococcus faecalis wild-type MTCC 2729. The proteins extracted from E. faecalis biofilms were subjected to 2-DE gel electrophoresis followed by Coomassie Brilliant Blue staining, in-gel digestion and matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) MS analysis. The study identified a total of 13 proteins, with the majority of the differentially expressed proteins belonging to the translation elongation machinery. This study also proposed that deficient biofilm formation of the strain is due to the underexpression of the osmotically inducible protein C, which is an OsmC/Ohr family oxidative stress protein. Somewhat similar observations were made in a label-free proteomics study on L. monocytogenes biofilm, which also employed both strong and weak biofilm formers, and compared their proteomics profiles [139]. The study suggested that the biofilm mode of growth is associated with an abundance of stress-defence proteins, which is related to the more chemically resistant phenotype compared to the planktonic mode. The strong biofilm former showed a higher level of resilience and had overall more stress tolerance than the weak biofilm former. Moreover, in a recent study, the proteins required for Streptococcus mutans biofilm formation in a competitive environment were analysed by a gel-based 2-DE proteomics approach. Proteins were extracted from the biofilms of S. mutans grown in the presence and the absence of S. gordonii, a competitive coloniser of the tooth surface [74]. The analysis identified that the peroxide-resistant protein Dpr is increased in the biofilms of S. mutans grown in the presence of S. gordonii. This shows that Dpr might be essential for the survival of S. mutans on teeth surfaces in the presence of other colonising oral streptococci.
Fibrinogen isoforms as potential blood-based biomarkers of Alzheimer’s disease using a proteomics approach
Published in International Journal of Neuroscience, 2022
Siti Hajar Rehiman, Siong Meng Lim, Fei Tieng Lim, Ai-Vyrn Chin, Maw Pin Tan, Shahrul Bahyah Kamaruzzaman, Kalavathy Ramasamy, Abu Bakar Abdul Majeed
The MS analysis was performed at the Medical Biotechnology Laboratory, University Malaya using the MALDI ToF/ToF 5800 mass spectrometry (Sciex, Framingham, USA). The instrument was first calibrated with trypsin-digested beta-galactosidase from Escherichia coli (Sciex, Framingham, USA). Internal control was prepared in-house using Coomassie-stained bovine serum albumin (5 µg/mL) gel plugs. In-gel digestion by trypsin was performed according to the protocol as described by Shevchenko, Tomas [43]. Complex proteins were first degraded enzymatically by trypsin at specific sites to generate peptides. Protein identification by MALDI ToF/ToF shows high sensitivity and accuracy exploiting peptide-mass fingerprinting (PMF) [44]. PMF refers to the matching of peptide mass obtained from the experiment against the theoretical peptide mass available in the protein databases [44]. The matching is performed via MASCOT (Matrix Science, London, UK).
Clinical improvement of ocular surface parameters in dry eye patients following treatment with urea/crosslinked-hyaluronate eyedrops correlates with the secretion of MUC-4
Published in Expert Review of Ophthalmology, 2021
Rosa Longo, Anna Avesani, Giulia Dalla Mura, Daniele Dell’Orco, Stefano Manfredini, Giacomo Panozzo
Proteins in the liquid tear milieu were separated by SDS-PAGE with a 15% polyacrylamide gel. The resulting gels were stained with silver staining technique (Merck, KGaA, Darmstadt, Germany), which has a higher sensitivity with respect to the more common Blue-Coomassie staining and it is a good means for processing poorly concentrated samples, to detect the protein in the low nanomolar range and it is compatible with subsequent protein digestion and mass spectrometry analysis. Protein bands were excised from the gels and subjected to an in-gel digestion protocol consisting of dithiothreitol reduction at 56°C (10 mM, 20 min) followed by iodoacetamide alkylation (55 mM, 20 min in a dark environment), and finally trypsin digestion (12.5 μg/μl) overnight at 37°C. The reaction was stopped by adding 0.1% trifluoroacetic acid (TFA-Merck). A piece of gel serving as a control was cut from a blank region and processed in parallel with the samples. After digestion, peptides were extracted with acetonitrile and trifluoroacetic acid solution by Zip-Tip C18 tips. Protein identification was carried out by MALDI-TOF-TOF.
Concepts and strategies of soybean seed proteomics using the shotgun proteomics approach
Published in Expert Review of Proteomics, 2019
Cheol Woo Min, Ravi Gupta, Ganesh Kumar Agrawal, Randeep Rakwal, Sun Tae Kim
Efficient protein digestion is critical to the successful identification of proteins. Typically, the solubilization of proteins using buffers that contain detergents (e.g. sodium dodecyl sulfate or urea) and the efficient elimination of the detergents from the samples are crucial steps in MS-based proteomic analysis. Two main digestion methods are used in gel-based and gel-free proteomics and are termed in-gel digestion and in-solution digestion, respectively. However, residual acrylamide in the products of in-gel digestion and residual salts and detergents in the products of in-solution digestion can reduce the efficiency of enzymatic digestion and peptide recovery. Therefore, researchers have developed an alternative approach, namely, filter-aided sample preparation (FASP) [55], in which digestion is performed in an ultrafiltration device that facilitates the removal of detergents and ensures efficient trypsin digestion. Indeed, this method was reported to increase the number of identified proteins by two-fold [56,57], which suggests that the method would be useful for increasing the proteome coverage of plant samples.