Post-Translational Modification PTM

In addition to the naturally occurring PTM, there may be alterations consequent to sample preparation, including those added intentionally, such as the reduction and alkylation of cysteines, or unintentionally, e.g., the oxidation of methionine. 

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In addition to the 20 common amino acids, some modified amino acids are also found in several proteins. These amino acids are normally altered via a process of post-translational modification (PTM) reactions (i.e. modified after protein synthesis is complete). Almost 200 such modified amino acids have been characterized to date. The more common such modifications are discussed separately in Section 2.5. 

Differences in post-translational modification (PTM) detail. Human therapeutic proteins produced in several recombinant systems (e.g. yeast-, plant- and insect-based systems Chapter 5) can display altered PTM detail, particularly in the context of glycosylation (Chapter 2). Some sugar residues/motifs characteristic of these systems can be highly immunogenic in humans. 

MS-Based Analysis of Post-Translational Modifications (PTMs)... 

The proteome is the set of expressed proteins at a given time under defined conditions it is dynamic and varies according to the cell type and functional state. One of the main differences when working with proteins is that there is not an amplification methodology for proteins comparable to PCR. Physical and chemical diversity of proteins are also higher than nucleic acids. They differ among individuals, cell types, and within the same cell depending on cell activity and state. In addition, there are hundreds of different types of post-translational modifications (PTMs), which evidently will influence chemical properties and functions of proteins. PTMs are key to the control and... 

The approaches described in the previous section enable the molecular-mass determination of intact proteins, generally with an accuracy better than 0.01%. Further stractural characterization of the protein requires determination of possible post-translational modifications (PTM) as well as the amino acid sequence. In addition, issues related to tertiary and quaternary stracture of the protein, the presence of cofactors, etc., may be relevant. LC-MS plays an important role in the primaiy and secondaiy stractural characterization of proteins, i.e., in terms of amino-acid sequencing and PTM. The procedure generally involves chemical or enzymatic treatment of the intact protein, acquisition of a peptide map or peptide mass fingerprint by either direct infusion (nano-)ESI-MS or RPLC-MS, and the amino-acid sequencing of individual peptides by means of product-ion MS-MS. Further experiments may be needed in relation to PTM, as outlined in more detail in Ch. 19. 

The science community has clearly established the essential role which protein and DNA sequences play in the understanding of biological systems. The sequences themselves are informative Indeed, many software tools are available which allow to make sense of the primary sequence information. Take those that analyse protein sequences for domains and active sites, perform similarity and homology searches, or predict the three-dimensional structure or physico-chemical parameters. However, raw sequences contain insufficient information, per se. One cannot infer any description or understanding of the level of expression of the active proteins, the content of post-translational modifications (PTMs), the tertiary structure and, what is perhaps the most relevant information, a protein s function. Like the sequences themselves, all these added value data need to be captured in various databases. These databases have to be queried by different types of users in proteomics and should therefore be easily searchable by software tools and be inter-linked in order to document the correspondences between the type of information provided by the different databases. 

Phosphorylation, principally on serine, threonine, or tyrosine residues, is one of the most important and abundant post-translational modifications (PTMs), with more than 30% of proteins being modified by the covalent attachment of one or more phosphate groups. It plays a critical role in the regulation of various cellular processes including cell cycle, growth, apoptosis, and transmitting extracellular signals to the nucleus. In fact, protein phosphorylation is... 

A biological membranes system is typically formed by the combination of lipids and proteins. In eukaryotic cells, the plasma membrane, also referred to as the cell membrane, is a protective barrier which regulates what enters and leaves the cell. The endomembrane system is composed of different kinds of membranes which divide the cell into structural and functional compartments within a eukaryotic cell, such as the endoplasmic reticulum, Golgi apparatus, mitochondria, endosome and lysosome. Covalent modification of proteins with lipid anchors (protein lipidation) facilitates association of the lipidated proteins with particular membranes in eukaryotic cells. Protein lipidation is one of the most important protein post-translational modifications (PTMs). Studying lipidated protein function in vitro or in vivo is of vital importance in biological research. 

A recent implementation of ETD reactions on the third type of hybrid instrument, a hybrid LIT/FT-ICR instrument [76], presents another example of the value in bringing high mass accuracy and mass-resolving power to the measurement of ion/ion reaction products. The hybrid instrument used a hexapole LIT as the reaction vessel by the superposition of auxiliary RF signals to the end lenses to effect mutual storage ion/ion reactions, the products of which were sent directly to the adjacent FT-ICR for mass analysis. The arrangement of the positive and negative ions sources is very similar to that for the Bruker Daltonics HCTultra post-translational modification (PTM) ion trap mass spectrometer [42] described in the previous section, which introduces positive ions formed by ESI from the front end of the instrument and... 

Electron-based dissociation methods (ETD and its forerunner electron capture dissociation, ECD) are highly efficient at generating c- and z -type fragment ions from peptide and whole protein precursor ions via a process that is essentially independent of peptide length, amino acid composition, and post-translation modification (PTM) state [28,45,76-81]. Precursor charge state and m/z-value are important factors, however, that can affect the dissociation efficiency of ETD methods [82-85]. 

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