Interactions between sea lice and their hosts
G. F. Wiegertjes, G. Flik in Host-Parasite Interactions, 2004
In the study by Valenzuela et al. (2002) proteins were identified by Edman degradation, a process that requires relatively large amounts of the protein. To obtain these proteins from small animals is often a very laborious and time-consuming process. Another common approach used for large-scale protein identification is proteomics. Proteomics allows the generation of amino acid sequence data from very small amounts of protein using mass spectrometry. In the past proteomics has been used to identify proteins that have been separated by one- or two-dimensional SDS-PAGE. More recently techniques have been developed to separate and identify proteins directly from biological extracts. Proteomic studies on sea lice are likely to be limited by the availability of protein sequences within available databases. As mentioned above conducting molecular studies of the parasite and using those data as a source of sequence information for protein identification may overcome this limitation.
Affinity Modification — Experimental Methods
Dmitri G. Knorre, Valentin V. Vlassov in Affinity Modification of Biopolymers, 1989
Some affinity reagents, in particular photoreactive ones, modify several amino acid residues within one peptide. An example can be the experiment on photoaffinity modification of mitochondrial F1ATPase with 2-azido [α-32P]adenosine diphosphate. The modified enzyme was cleaved to small peptides and the peptide containing radioactivity was isolated, identified on the basis of amino acid composition and N-terminal amino acid, and subjected to Edman degradation. It was found that the label was localized on four amino acids (Figure 6).332 In the cases cited above, Edman degradation allowed the modified amino acid residues to be identified precisely. It should be noted that it is not always possible. Some cases have been reported where sequencing the modified peptides using Edman degradation was complicated. Thus, when modified peptides isolated from myosin treated with N-(4-azido-2- nitrophenyl)-2-aminoethyl diphosphate were sequenced, some radioactivity was released at each cycle. The most reasonable explanation was that the linkage of the [3H]-labeled reagent with the modified residue was acid-labile and the reagent grouping was released each cycle during the acid cleavage steps of the Edman procedure.333 Analysis of the peptides isolated from E. coli ADP-glucose synthetase modified by 8-azido-ADP[14C] glucose could not be accomplished due to the lability of the glucosidic linkage at acidic pH.334
Mechanisms of Fibril Formation and Cellular Response
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin in XIth International Symposium on Amyloidosis, 2007
Isolation and Characterization of Amyloid Protein: The rectal tumor biopsy was hand homogenized in 0.1 M sodium citrate, 0.15 M sodium chloride and centrifuged twice. The final pellet was solubilized in 8 M guanidine hydrochloride containing dithiothreitol, alkylated with iodoacetic acid, centrifuged and the supernatant was fractionated on a Sepharose CL6B column (1,2). Fractions from approximately the second half of the void volume to column volume area were pooled, exhaustively dialyzed against water, and lyophilized. The lyophilized material was suspended in 10% acetic acid and portions used for further analysis. Samples were analyzed by SDS-PAGE using a Tris-Tricine system and electrophoretically transferred to polyvinylidine difluoride membrane for Edman sequence analysis. A portion was digested with trypsin and the resulting peptides were fractionated by reverse phase HPLC on a Beckman Ultrasphere ODS column using an acetonitrile gradient in 0.1% trifluoroacetic acid in water. Edman degradation analyses were
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).
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.
Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray
Published in mAbs, 2019
Yuan Mao, Lichao Zhang, Andrew Kleinberg, Qiangwei Xia, Thomas J. Daly, Ning Li
Several analytical techniques have been used to confirm the protein sequence of therapeutic monoclonal antibodies. One such technique is N-terminal protein sequencing by Edman degradation, which has been used in the pharmaceutical industry for decades to confirm monoclonal antibody identity.6 Edman degradation is still considered the most reliable technology, although it does have several disadvantages. Sample preparation for Edman degradation is tedious and consumes significant amounts of chemical reagents. The sequencing step in this technology is highly automated, but the method is low-throughput and can usually only sequence one amino acid residue per hour.7 The efficiency of Edman degradation after 50 residues is low, making it unsuitable for sequencing large molecules such as monoclonal antibodies, which have more than 1200 amino acid residues.8
Related Knowledge Centers
- Acid
- Chromatography
- Peptide
- Peptide Bond
- Protein Sequencing
- Residue
- Amino Acid
- Phenyl Isothiocyanate
- Electrophoresis
- N-Terminus