Post -translational

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

While electrospray is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct measurement of molecular mass on proteins has been difficult, with the obtained values accurate to only tens or even hundreds of Daltons. The advent of electrospray means that molecular masses of 20,000 Da and more can be measured with unprecedented accuracy (Figure 40.6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g., glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. 

Post-translational modifications to proteins are biochemical in origin and alter the measured molecular mass relative to that calculated for an untranslated sequence. 

Biosynthesis. The biosynthesis of neuropeptides is much more complex and involves the multistep process of transcription of specific mRNA from specific genes, formation of a high molecular weight protein product by translation, post-translational processing of the protein precursor to allow for... 

Yeast. The advantages of expression in yeast include potentially high level production of proteins, the abiUty to have expressed proteins secreted into the media for ease of purification, and relatively low cost, easy scale-up. A disadvantage is that plasmid instabiUty may be a problem which can lead to low product yield. Whereas post-translational modification occurs in yeast, proteins are quite often hyperglycosylated. This is generally a problem with expression in Saccharomyces cerevisiae but not for the more recently used yeast host Pichiapastoris (25) (see Yeasts). 

Insect Cells. In this system the cDNA is inserted into the genome of an insect vims, baculovims. Insect cells, or Hve insect larvae, are then infected with the vims. In this way advantage is taken of the vims s natural machinery for repHcation utilizing the insect cell. This is one of the best systems available for high level production of native protein having post-translational modifications similar to those seen in mammalian cells. Disadvantages of this system include lytic—batch variations, comparatively slow growth, and cosdy scale-up. 

MammaBan. For mammalian proteins, mammalian cells offer the most natural host for expression. Problems of incorrect processing and post-translational modification are avoided using these cells. Mammalian cells are usually grown in continuous cell culture, reducing the variabiUty in results (see Cell CULTURE technology). Moderate-level production of native protein is possible. The procedure, however, is slow and very cosdy, and the level of protein expression is low. Thus large-scale production of proteins in mammalian cells is not practical. When low quantities of protein are sufficient, this system offers the several advantages described. 

ChEs present a wide molecular diversity that modulates their function in cholinergic synapses and non-synaptic contexts. This diversity arises at the genetic, post-transcriptional and post-translational levels. 

Both ChEs undergo several post-translational modifications, including glycosylation and glycosylphosphatidy-linositolation (GPI), phosphorylation and carbamylation. 

Glycosydphosphatidylinositolation The GlycoPho-sphatidyl Inositol moiety anchor of AChE consists exclusively of diacyl molecular species. Over 85% of the molecular species are composed of palmitoyl, stearoyl and oleoyl. The post-translational process of glypiation takes place in the endoplasmic reticulum, after completion of the polypeptide chain the newly synthesized protein interacts with a transamidase... 

Myristoylation is the post-translational addition of the 14-carbon fatty acid myristate to the N-terminal glycine of proteins via an amide link. Myristoylation of proteins helps to anchor them to membranes. 

Palmitoylation is the post-translational lipid modification of cysteine-residues in a variety of proteins. 

PPCs Prenylation is the post-translational addition of 15- or 20-carbon isoprenyl lipids to the C-terminus of proteins. Prenylation is an irreverable modification that anchors proteins to the membrane fraction of cells. 

After their synthesis (translation), most proteins go through a maturation process, called post-translational modification that affects their activity. One common post-translational modification of proteins is phosphorylation. Two functional classes of enzymes mediate this reversible process protein kinases add phosphate groups to hydroxyl groups of serine, threonine and tyrosine in their substrate, while protein phosphatases remove phosphate groups. The phosphate-linking... 

Proteosomal degration is the process by which improperly folded proteins or proteins with altered post-translational modifications are removed from a cell before they have a detrimental effect on cellular function. This is performed in small organelles known as proteosomes. Proteins are targeted for destruction in the proteosome by having a number of small ubiquitin molecules added. 

All small GTPases (except Ran) are post-translationally modified. Most important is the isoprenylation of the C-terminus. The type of modification is determined by... 

Poor Metabolizer Phenotype Population Pharmacokinetics Positron Emission Tomography Post-translational Modification Potassium Channels Potassium Competitive Acid Blockers PP... 

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