Analysis of Intact Protein Isoforms by Mass Spectrometry

Tipton, Jeremiah D.; Tran, John; Catherman, Adam D.; Ahlf, Dorothy R.; Durbin, Kenneth R.; Kelleher, Neil L.

doi:10.1074/jbc.r111.239442

Cited by 95 publications

(70 citation statements)

References 100 publications

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“…The techniques available thus far are useful for determining the stoichiometry of the entire proteome, but methodologies can also be developed for a smaller-scale studies. Another opportunity would be to explore "top-down" intact mass spectrometry (139) to study the acetylation of single intact proteins. This possibility would reveal site-specific stoichiometry and also paint a picture of the different species made up of combinations of acetylated residues on multiply acetylated proteins.…”

Section: Perspectivesmentioning

confidence: 99%

Regulation, Function, and Detection of Protein Acetylation in Bacteria

2017

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N -Lysine acetylation is now recognized as an abundant posttranslational modification (PTM) that influences many essential biological pathways. Advancements in mass spectrometry-based proteomics have led to the discovery that bacteria contain hundreds of acetylated proteins, contrary to the prior notion of acetylation events being rare in bacteria. Although the mechanisms that regulate protein acetylation are still not fully defined, it is understood that this modification is finely tuned via both enzymatic and nonenzymatic mechanisms. The opposing actions of Gcn5-related N-acetyltransferases (GNATs) and deacetylases, including sirtuins, provide the enzymatic control of lysine acetylation. A nonenzymatic mechanism of acetylation has also been demonstrated and proven to be prominent in bacteria, as well as in mitochondria. The functional consequences of the vast majority of the identified acetylation sites remain unknown. From studies in mammalian systems, acetylation of critical lysine residues was shown to impact protein function by altering its structure, subcellular localization, and interactions. It is becoming apparent that the same diversity of functions can be found in bacteria. Here, we review current knowledge of the mechanisms and the functional consequences of acetylation in bacteria. Additionally, we discuss the methods available for detecting acetylation sites, including quantitative mass spectrometry-based methods, which promise to promote this field of research. We conclude with possible future directions and broader implications of the study of protein acetylation in bacteria.

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Section: Perspectivesmentioning

confidence: 99%

Regulation, Function, and Detection of Protein Acetylation in Bacteria

2017

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“…As such, the conjugation of ChroP with "Bottom-up" MS analysis permits a partial assessment of the combinatorial aspect of the histone code. We envisage that the implementation of alternative MS strategies, such as "Middle-and Top-Down" for hPTMs mapping on longer peptides (>20 aa) up to on intact proteins [38][39][40] , will overcome this restraint.…”

Section: Discussionmentioning

confidence: 99%

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Soldi

Bonaldi

2014

JoVE

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Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locusspecific structural and functional configurations. Video LinkThe video component of this article can be found at

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“…Protein biotherapeutic catabolites can be investigated by a number of different types of ion sources [e.g., electrospray ionization, matrix-assisted laser desorption ionization, surface-enhanced laser desorption ionization (SELDI)] and mass analyzers [e.g., quadrupole, time of flight (TOF), ion trap/orbitrap]. Since the parameters for mass spectrometer choice are essentially the same as for general proteomic studies and protein analysis, the details will not be described here, as there are already excellent review articles covering this topic (Mann et al, 2001;Ens and Standing, 2005;Domon and Aebersold, 2006;Ahmed 2008;Tipton et al, 2011). Catabolites and other biotransformed entities can be confirmed by mass differences in the resultant mass spectra and corroborated by gas-phase fragmentation data if necessary.…”

Section: Workflows For Studying Protein Biotherapeutic Biotransformationmentioning

confidence: 99%

Biotransformation and In Vivo Stability of Protein Biotherapeutics: Impact on Candidate Selection and Pharmacokinetic Profiling

Hall

2014

Drug Metab Dispos

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Historically, since the metabolism of administered peptide/protein drugs ("biotherapeutics") has been expected to undergo predictable pathways similar to endogenous proteins, comprehensive biotherapeutic metabolism studies have not been widely reported in the literature. However, since biotherapeutics have rapidly evolved into an impressive array of eclectic modalities, there has been a shift toward understanding the impact of metabolism on biotherapeutic development. For biotherapeutics containing non-native chemical linkers and other moieties besides natural amino acids, metabolism studies are critical as these moieties may impart undesired toxicology. For biotherapeutics that are composed solely of natural amino acids, where end-stage peptide and amino acid catabolites do not generally pose toxicity concerns, the understanding of biotherapeutic biotransformation, defined as in vivo modifications such as peripherally generated intermediate circulating catabolites prior to end-stage degradation or elimination, may impact in vivo stability and potency/clearance. As of yet, there are no harmonized methodologies for understanding biotherapeutic biotransformation and its impact on drug development, nor is there clear guidance from regulatory agencies on how and when these studies should be conducted. This review provides an update on biotherapeutic biotransformation studies and an overview of lessons learned, tools that have been developed, and suggestions of approaches to address issues. Biotherapeutic biotransformation studies, especially for certain modalities, should be implemented at an early stage of development to 1) understand the impact on potency/clearance, 2) select the most stable candidates or direct protein re-engineering efforts, and 3) select the best bioanalytical technique(s) for proper drug quantification and subsequent pharmacokinetic profiling and exposure/response assessment.

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Analysis of Intact Protein Isoforms by Mass Spectrometry

Cited by 95 publications

References 100 publications

Regulation, Function, and Detection of Protein Acetylation in Bacteria

Regulation, Function, and Detection of Protein Acetylation in Bacteria

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Biotransformation and In Vivo Stability of Protein Biotherapeutics: Impact on Candidate Selection and Pharmacokinetic Profiling

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