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Molecular & Cellular Proteomics 6:S20, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstracts

Session 2

Deciphering the Dynamics of Proteasome Interacting Proteins Using Quantitative Mass Spectrometry

X. Wang and L. Huang

Departments of Physiology and Biophysics and Developmental and Cell Biology, University of California, Irvine, CA

Protein-protein interactions are one of the major mechanisms for controlling protein functions in various cellular processes. In order to fully understand these functions, global mapping of protein-protein interactions has become a major goal in current proteomics research. In combination with affinity purification, mass spectrometry-based interactive proteomics has become the method of choice for analyzing functional protein complexes. In addition to the interactions of varied affinity, the dynamics of protein interactions is another important aspect for understanding the functions of protein interactions. The dynamics can be classified into two layers: dynamic protein interactions that change temporally with cellular states at different time of extracellular signaling, and those that have high association and dissociation (i.e. on/off) rates with their interacting partners. Although much effort has been made to understand the first layer of interaction dynamics, lack of an efficient strategy to distinguish stable and dynamic interactors has hampered the efforts towards the understanding of the second type. In this work, we have developed a new method, MAP (mixing after purification)-SILAC to quantitatively investigate the interactions of protein complexes by mass spectrometry. In combination with the original SILAC approach, stable and dynamic components are unambiguously distinguished based on their relative abundance ratio changes. We applied the newly developed strategies to decipher the dynamics of the 26S proteasome interacting proteins (PIPs), and have identified new putative PIPs while the nature of the identified interactors was fully characterized. The methods reported here provide a valuable expansion of proteomics technologies for identification of important but previously unidentifiable interacting proteins.

2.2

The Proteome Biology of Proteasome Complexes: Molecular Organization, Function, and Regulation

P. Ping

Department of Physiology and Medicine/Cardiology, NHLBI PPG on Myocardial Ischemia Injury, Proteomic Core Laboratory at CVRL, University of California, School of Medicine, Los Angeles, CA

The proteasome system plays a critical role in governing the intracellular protein degradation processes in mammalian cells. The proteome biology of these proteasome complexes, i.e., the assembly of proteasome subunits, the post-translational modifications of the subunits, and the associating partners of the complexes, dictates the intracellular proteolytic functions that these multi-protein complexes perform. Using an organelle proteomic approach, our recent investigations have defined the molecular organization, function, and regulation of this organelle in mammalian cell types.

2.3

Identification of Newly Synthesized Proteins Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT).

D. C. Dieterich¹, A. J. Link², D. A. Tirrell², J. Graumann¹, and E. M. Schuman¹

Division of ¹Biology/HHMI and ²Chemistry, Caltech, Pasadena, CA

Alterations in protein synthesis and degradation enable cells, including neurons, to adapt to changing external conditions. In neurons, there is increasing evidence that local dendritic protein synthesis is used to allow individual synapses to respond dynamically to the environmental changes that accompany the establishment, maintenance and plasticity of synaptic connections. The identification of the activity-modulated dendritic proteome promises to offer a more thorough understanding of synaptic plasticity at the molecular level. To isolate and identify dendritically synthesized proteins we have developed a new protein tagging strategy that can be used in combination with mass spectrometry. The protein tagging is based on an azide-alkyne ligation using the azide-group bearing non-canonical amino acid azidohomoalanine (AHA), which serves as a surrogate for methionine. Proteins bearing AHA can subsequently be tagged with an alkyne-bearing affinity tag. After tryptic digestion of affinity-purified proteins, mass spectral analysis is achieved by utilizing MudPIT (Multidimensional Protein Identification Technology) followed by bioinformatical analysis. Initial experiments show, that AHA can be incorporated into newly synthesized proteins of HEK293 cells and cultured hippocampal neurons. In control experiments where AHA was replaced with methionine, no biotinylated proteins were recovered following avidin-chromatography. In tandem mass spectrometry analysis of avidin-purified proteins from AHA (2 hr)-treated whole cell lysates of HEK293 more than 190 proteins, including an overexpressed control protein, were identified. To identify the dendritic proteome, newly synthesized proteins from either rat brain synaptoneurosomes or isolated dendrites of hippocampal cultures are analyzed.

D.C.D. was supported by the German Academy for Natural Scientists LEOPOLDINA (BMBF-LPD9901/8-95).

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This Article Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Search for Related Content Social Bookmarking                      What's this?