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Methods for Sequence Determination
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Procedures for sequence analysis by the Edman degradation fall into two major categories differing in the type of method used for determination of the amino acid at each step. In so-called indirect Edman procedures, a portion of the sample is removed at the beginning and after each cycle and analyzed either quantitatively for total amino acid composition or qualitatively for the identity of the NH2-terminal. In direct methods, the thiazolinone produced at each step is extracted, converted to the more stable thiohydantoin (Reaction 3), and identified. Manual procedures of both types are used; automated sequenators have generally used direct identification.
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
for extraction, which may be quite limited in the case of fine-needle biopsy specimens that generally are only ~1 X 15 mm in size (based on present technology, ~1-10 pmol of extracted protein is required). It should be noted that if instrumentation lacks de novo sequencing capabilities, it is necessary that the database include pertinent algorithms and mass data, which may not be available for novel amyloidogenic proteins. In such instances, amino acid sequencing by Edman degradation of enzymatically or chemically derived peptides is required. Furthermore, it must be recognized that extracts, especially those obtained from fresh (unfixed) tissue, often contain nonfibrillar intracellular or extracellular proteins that are co-deposited with the amyloid, but do not represent amyloidogenic precursor molecules.
Serum Amyloid P Component in Mink *
Published in Gilles Grateau, Robert A. Kyle, Martha Skinner, Amyloid and Amyloidosis, 2004
Lone A. Omtvedt, Tale N. Wien, Theresa Myran, Knut Sletten, Gunnar Husby
Isolation and characterisation of serum pentraxins; Proteins were purified from mink serum by affinity chromatography, either using PE coupled ECH-sepharose 4B (Amersham Biosciences) or immobilised PC-gel (Pierce Biotechnology) (2). The purity of the sample was analysed by SDS-PAGE and N-terminal sequencing. Alkylated and non-alkylated SAP purified from control serum, was digested with trypsin, with staphylococcal V8 protease or with endoproteinase ASP-N (Roch) according to the manufacturer (7–8). Chemical fragmentation with CNBr was performed both on filter and in solution (9). Proteins and peptides were analysed by automatic Edman degradation and by mass spectrometry.
Twenty years of proteomics in radiation biology – a look back
Published in International Journal of Radiation Biology, 2022
The gel-based separation of proteins was as such not a new method. I remember running the so called O'Farrell gels during my PhD in Sweden in early 1980's but after moving to Germany for my post-doc I realized that this name for 2D-SDS-PAGE gels was not popular there. Actually, O'Farrell and Klose discovered and published this method at the same time in the spring of 1975 (Klose 1975; O'Farrell 1975). The problem with those first gels was that one could discover changes in the protein expression or even mutations by looking at the gels (you had to rely on your eyes since there was no software available at that time) but it was not possible to identify the proteins. Edman degradation that was mainly used for purified proteins was tedious and slow. First the developments of mass spectrometry technologies such as matrix-assisted laser desorption/ionization (MALDI) (Hillenkamp et al. 1991) and electrospray ionization (ESI) (Whitehouse et al. 1985) meant a breakthrough. By these methods one could identify the peptides (and so the proteins they originate from) by measuring the velocity of the peptide ions in a magnetic field that in its turn depended on the mass-to-charge ratio, the so called time-of-flight (TOF) (Mirsaleh-Kohan et al. 2008).
Developments with bead-based screening for novel drug discovery
Published in Expert Opinion on Drug Discovery, 2019
In summary, most of the technical challenges associated with first-generation OBOC libraries have now been resolved. Small molecules and other compounds that cannot be directly sequenced by Edman degradation or tandem mass spectrometry can now be prepared and screened against intended targets by encoding them with linear peptides or DNA tags. Spatially segregated beads may be used to confine the encoding tags to the bead interior, preventing them from interfering with library screening. They also permit the synthesis of OBOC libraries with reduced ligand density at the bead surface, increasing the stringency of library screening while largely eliminating nonspecific binding caused by the avidity effect. Sensitive assays have been developed to quantitatively determine the binding affinity of initial hits to the intended target in the solution phase, by using compounds released from single 90-μm beads. This eliminates the expensive step of individual resynthesis and testing of a large number of initial hits. Finally, a number of automation technologies (e.g. flow cytometry and magnetic sorting) enable ultra-high-throughput screening of millions of OBOC beads/compounds very rapidly and inexpensively. These technological advancements have recently led to a renaissance of OBOC libraries in chemical biology and drug discovery.
A Peptide from Kiwifruit Exerts Anti-Inflammatory Effects in Celiac Disease Mucosa
Published in Journal of the American College of Nutrition, 2019
Ilaria Russo, Chiara Del Giorno, Ivana Giangrieco, Najla Hajji, Maria Antonietta Ciardiello, Paola Iovino, Carolina Ciacci
The peptide preparation was considered pure and therefore suitable for the functional studies when it appeared free of any contaminant molecule. The purity of the peptide preparation was analyzed by RP-HPLC and N-terminal amino acid sequencing. The RP-HPLC separation showed a single peak in the chromatographic profile, thus indicating the absence of contaminant molecules. Amino acid sequencing of the N-terminal region of the purified kissper was performed with an Applied Biosystems Procise 492 automatic sequencer (Applied Biosystems, Foster City, CA), equipped with online detection of phenylthiohydantoin amino acids (16). Briefly, a peptide solution at the concentration of 1 mg/ml was diluted 1:1 with distilled water. Four microliters, containing 2 µg of kissper, was picked up from the 0.5 mg/ml peptide solution and loaded on the automated amino acid sequencer. Ten sequencing cycles, based on Edman degradation chemistry, were run and the following amino acid sequence was obtained: ISSCNGPCRD. This sequence allowed the unequivocal identification of kissper. In fact, the homology search performed using the sequence ISSCNGPCRD and the BLAST algorithm, on the expasy website www.expasy.org), showed that the only kiwifruit protein molecule having this N-terminal sequence was kissper. In addition, the results obtained from the automated sequencing showed the absence of contaminant amino acids. Hence, they also confirmed the purity of kissper preparation.