And that's why Edmund degradation procedures sometimes called end terminal peptide sequencing. Now, the Edmund Degradation Procedure is really a cycle of three different chemical reactions, and these three reactions remove one and terminal amino acid residue at a time and then identifies that in terminal amino acid residue upon removal and because it is the end terminal amino acid residue that's removed one at a time, the peptide is actually sequence from the n terminal end towards the C terminal end. But for now, all I want you guys to know is that the Edmund degradation procedure can only be used on one single small peptide chain at a time. Later, in our course, when we talk about Edmund degradation reaction efficiency, we'll explain exactly why the peptide needs to be small. And so this peptide actually needs to be relatively small now. Which means that before Edmund Degradation, we're going to need to use our protein purification techniques toe isolate one particular peptide of interest. And so the Edmund Degradation procedure can Onley be used on one single poly peptide chain at a time. So the Edmund Degradation procedure is really just a protein sequencing technique developed by a scientist named Pair Edmund way back in the sixties. The aim of this chapter is to introduce to basic theory, practical applications and limitations of the various methods, to enable the non-expert scientist to decide which method is best suited for his project and which kind of sample preparation is necessary.In this video, we're going to talk about Edmund Degradation. De novo sequencing by MS of peptides is possible, but very time consuming and not a routine application, in contrast to Edman degradation. This approach requires that the protein is known and listed in the database. Proteins are either identified by searching databases with the masses of proteolytic peptides (peptide mass fingerprinting) or using fragmentation data (raw MS/MS spectra or sequence tags). They can be combined with a variety of mass analyzers (TOF, quadrupole, ion trap). These are MALDI (matrix assisted laser desorption and ionization) and ESI (electrospray ionization). Two different ionization methods are commonly used to generate peptide/protein ions for MS analysis. It is the method of choice for sensitive analysis and large-scale applications (proteomics). MS is a very fast method that can be automated. In contrast to Edman degradation, complex mixtures such as tryptic digests can be analyzed, making HPLC separation of peptides unnecessary. MS has routinely been used with peptides in the range of 100 fmol or even less. It is also the method of choice for sequencing unknown proteins/ peptides and modified peptides. Nevertheless, Edman degradation is still the workhorse in the lab for routine work such as identification of blotted proteins. Sequencing of a peptide requires many hours, the sensitivity is in the range of 2-5 pmol of a purified peptide. Edman degradation is the older method based on successive removal of N-terminal amino acids by chemical methods. Structural analysis can be done by Edman degradation or mass spectrometry (MS). The separation method of choice is gel electrophoresis, followed by blotting to PVDF membrane for N-terminal sequencing or by in-gel digestion to generate peptides that can be separated by HPLC. Proteins of interest are typically available in low microgram amounts or even less. Nevertheless, protein sequencing/identification is still indispensable to analyze the proteins expressed in a cell, to identify specific proteins, and to determine posttranslational modifications. The primary structure of proteins is nowadays determined by DNA sequencing, and a variety of genomes are already known.
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