Protein posttranslational modifications PTMs

The protein posttranslational modifications (PTMs) play a crucial role in modifying the end product of expression and contribute towards biological processes and diseased conditions. Important posttranslational modifications include phosphorylation, acetylation, glycosylation, ubiquitination, and nitration [Mann and Jensen, 2003], The analysis of posttranslational modifications on a proteome scale is still considered an analytical challenge [Zhou et al., 2001] because of the extremely low abundance of modified proteins among very complex proteome samples. 

Knowledge of protein primary sequence, quantities, posttranslational modifications (PTMs), structures, protein-protein (P-P) interactions, cellular spatial relationships, and functions are seven important attributes (see Table 4.2) needed for comprehensive protein expression analysis. It is this multifold and complex nature of protein attributes that has spawned the development of so different many proteomic technologies. Some of these challenges in proteomic analysis include defining the identities and quantities of an entire proteome in a particular spatial location (i.e., serum, liver mitochondria, brain), the existence of multiple protein forms and complexes, the evolving structural and functional annotations of the human and rodent... 

PTMs Posttranslational modifications (PTMs) are the addition of chemical structures to -R groups of proteins. PTMs also include other processing of proteins such as proteolytic removal of chemical groups like phosphates by phosphatases or sugars by glycosidases or also of primary sequence by proteases. 

DNA has a form of double helix and contains nearly 3 billion base pairs, each of which consists of one of four types of nucleotides such as adenine, thymine, cytosine, and guanine. The human genome, the complete human DNA, would be equivalent to about 250 volumes of Manhattan phone directories when it is printed. Figure B.l illustrates the processes associated with protein synthesis transcription, translation, posttranslation modification (PTM), and folding. 

Phosphorylation, or the attachment of a phosphate group to amino acid side chains, is one of the most abundant posttranslational modifications (PTMs) of proteins. Phosphorylation reactions are mediated by phosphotransferase enzymes, termed kinases, with ATP as the typical source of the transferred phosphoryl group. Ser, Thr, and Tyr are the most commonly phosphorylated residues in eukaryotes, while His and Asp phosphorylation has also been observed, predominantly in prokaryotes. Protein activity, localization, and structure as well as protein-protein interactions are all affected by protein phosphorylation [1, 2]. As kinases play integral roles in cellular signaling, dysregulated kinase function has emerged as a driver for many different disease states, including... 

Posttranslational modification (PTM) with functional groups is a universal mechanism for diversifying the activities of proteins. PTMs can affect many properties of proteins, such as localization, activity status, interaction networks, solubility, folding, turnover, or stabUity. It is therefore of vital importance to accurately determine the identities of modified proteins, the modified amino acid residues, and the covalently attached group. This chapter describes the process of PTM identification using the adenylylation (i.e., the covalent transfer of an adenosine monophosphate (AMP)) of rat sarcoma related in brain (Rab) proteins hy Legionella pneumophila enzymes as an example. It also deals with the development of PTM-specific antibodies from synthetic peptides. This account underlines the importance of chemical biology in the elucidation of PTMs. 

Top-down proteomics [1] is the identification and characterization of a mature, intact protein (or proteins) using primarily mass spectrometry (MS)-based techniques and the sequence information contained in genomic/proteomic databases. Unhke bottom-up proteomics [2-4], where a protein (or proteins) is digested into peptides prior to MS or tandem mass spectrometry (MS/MS) analysis [3,4], the top-down approach involves measuring the molecular weight (MW) of the intact, mature protein with its associated posttranslational modifications (PTMs) if any. The intact protein ion is then fragmented in the gas phase in order to determine its amino acid sequence as well as the location and identification of PTMs. From its earliest development, top-down proteomics has been primarily the domain of electrospray ionization (ESI) [5] (which generates mul-... 

Microbial organisms in several instances are not suitable for the production of large therapeutic proteins. Most human proteins require, for activity, glycosylation and other posttranslational modifications (PTM) that bacteria are unable to perform correctly. Mammalian cell lines are the best expression systems for these products. Most recombinant proteins belong to the WHO... 

In another application, UHPLC-MS technology was developed for rapid comparison of a candidate biosimilar to an innovator monoclonal antibody (mAb) (37). In this study, UHPLC-MS was developed for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for mAb products. Although the biosimilar product is expected to have the same amino acid sequence and modifications as the innovator s product, the observed intact mass by UHPLC-MS was different for the biosimilar compared to the innovator protein. Peptide mapping using UHPLC-MS/MS (38) revealed that the mass difference between the biosimilar and the innovator s product was due to a two amino acid residue variance in the heavy chain sequence of the biosimilar (Figure 8.6). 

We have come a long way in the journey toward understanding the molecular basis of life. Half a century ago, Francis Crick proposed the central dogma as a working model that credits the three-dimensional structure and function of proteins to their one-dimensional progenitors DNAs and RNAs [38], However, the discovery of posttranslational modification (PTM) makes it clear that the structure and function of proteins and polypeptides can be dictated by factors other than their encoding nucleic acids and amino acid building blocks [39]. 

Biological samples that are useful for clinical diagnosis often contain very complex mixtures of proteins, as up to a few thousand may be present in tissue extract or body fluids, and each of them can have a variety of posttranslation-al modifications (PTMs). Before the analysis of individual proteins, an essential first step in proteomics is carrying out multidimensional separations to fractionate the sample into its individual constituents. Fractionations are needed to obtain high analytic sensitivity, which is done by increasing the relative concentration and purity of the individual components (as discussed in Section 5). To facilitate the use of small sample sizes and to prevent loss of... 

Figure 4.13. Summary of advantages and limitations of representative proteomic platforms for protein expression analysis. The capabilities and advantages (solid line) are compared to the drawbacks (dotted lines) for each proteomic platform. Explanation of advantages and limitations are briefly highlighted here and more thoroughly discussed in the text. Abbreviations for each platform are provided in the Figure 4.1 legend and in the text. Other abbreviations used are PTMs, posttranslational modifications ID, identification.

Peptide sequence analysis (PSA) of the peptides that contains a PTM. If the amino-acid sequence of the protein is not known or the position is not unambiguously determined in PMF, sequence analysis of the modified peptides is necessary. Strategies for shotgun identification (Ch. 18.3.2 ) of posttranslation modifications are reviewed by Cantin and Yates [4]. 

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