Mass Spectrometric Characterization of the Affinity-Purified Human 26S Proteasome Complex

Wang, Xiaorong; Chen, Chi-Fen; Baker, Peter R.; Chen, Phang-Lang; Kaiser, Peter; Huang, Lan

doi:10.1021/bi061994u

Cited by 248 publications

(298 citation statements)

References 43 publications

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“…LC-MS/MS analysis has also been previously described in conjunction with affinity purification to identify proteins and associated modifications using stably expressed protein in cell lines. 34 The use of affinity chromatography for both analyte enrichment and purification in the measurement approach can be a potential source of measurement bias and must be properly evaluated. If the monoclonal antibody is differentially selective for specific molecular forms of CRP, or if there are matrix effects that prevent equilibrium binding of CRP to the antibody, the amount of CRP captured for quantification after affinity chromatography will not be well correlated to the actual amount in the serum.…”

Section: Discussionmentioning

confidence: 99%

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Kilpatrick

Bunk

2009

Anal. Chem.

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This paper describes the development of a reference measurement procedure to quantify human C-reactive protein (CRP) in serum using affinity techniques prior to tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the certification of reference materials in clinically relevant ranges. The absence of a suitable internal standard for the CRP measurement, necessary to eliminate potential measurement bias in both the affinity purification and trypsin digestion steps, was addressed using the method of standard addition. The standard addition quantification approach was combined with affinity purification, using an anti-CRP monoclonal antibody conjugated to polystyrene beads, trypsin digestion of the purified protein, and LC-MS/MS analysis of CRP tryptic peptides. The effectiveness of intact protein affinity purification was evaluated through the measurement of CRP in several serum-based CRP control materials, yielding levels that were comparable to their expected mean concentration values. Quantitative results were confirmed with an external calibration approach. This study demonstrates the feasibility of affinity purification with LC-MS/MS for the reference measurement procedure development of low abundance serum protein analytes.Reference measurement procedures are used for a variety of purposes including (1) the assessment of the measurement quality of routine measurement procedures, (2) the value assignment of high-order calibration solutions (the so-called "master calibrators") for routine assays, and (3) the value assignment of certified reference materials. 1,2 In these roles, reference measurement procedures are considered "higher-order" measurement procedures, as they aim to achieve a higher level of accuracy and precision than the routine assay for the analyte being measured, producing a value with a low uncertainty of defined magnitude. In order for a reference measurement procedure to be fit for this purpose, it must be thoroughly evaluated to identify sources of bias and assess their magnitude. [3][4][5] Minimizing or eliminating bias is necessary to measure the most accurate, true concentration of the analyte. Because of the need for high levels of accuracy and precision, reference measurement procedures are typically low-throughput, labor intensive, and often costly to perform, in sharp contrast to what is desirable for routine measurements.Reference measurement procedures are key elements in the establishment of measurement standardization and metrological traceability in clinical chemistry. For more than 30 years, isotope dilution mass spectrometry (IDMS) has been one of the analytical techniques used frequently in reference measurement procedures, particularly for small molecule and inorganic analytes of clinical importance. 6 Despite this, few reference measurement procedures exist for clinically relevant proteins. This is one reason why so many clinical protein analytes are measured in arbitrary International Units (IUs); without a reference measurement procedure to...

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Section: Discussionmentioning

confidence: 99%

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Kilpatrick

Bunk

2009

Anal. Chem.

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“…Human proteasomes were affinity-purified on a large scale from a stable HEK293 cell line (30). Cryo-EM data were collected with the supercounting mode of a Gatan K2 Summit direct electron detector installed on an FEI Tecnai Arctica microscope operating at a nominal magnification of 21,000× and an acceleration voltage of 200 kV.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for dynamic regulation of the human 26S proteasome

Chen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

159

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The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

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“…After mixing, affinity purification was carried out by binding to streptavidin resin followed by TEV elution (16). In addition to the proteasome complexes, their interacting partners that survive during the purification will be captured.…”

Section: Pam (Purification After Mixing)-silac and Map (Mixing After mentioning

confidence: 99%

“…Cell lines were grown for more than seven cell doublings in the labeling media to ensure complete labeling. The cells were then grown to confluence prior to cell lysis as described previously (16).…”

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confidence: 99%

Identifying Dynamic Interactors of Protein Complexes by Quantitative Mass Spectrometry

Wang

Huang

2008

Molecular & Cellular Proteomics

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184

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Dynamically interacting proteins associate and dissociate with their binding partners at high on/off rates. Although their identification is of great significance to proteomics research, lack of an efficient strategy to distinguish stable and dynamic interactors has hampered the efforts toward this goal. In this work, we developed a new method, MAP (mixing after purification)-SILAC (stable isotope labeling of amino acids in cell culture), to quantitatively investigate the interactions of protein complexes by mass spectrometry. In combination with the original SILAC approach, stable and dynamic components were effectively distinguished by the differences in their relative abundance ratio changes. We applied the newly developed strategies to decipher the dynamics of the human 26 S proteasomeinteracting proteins. A total of 67 putative human proteasome-interacting proteins were identified by the MAP-SILAC method among which 14 proteins would have been misidentified as background proteins due to low relative abundance ratios in standard SILAC experiments and 57 proteins have not been reported previously. In addition, 35 of the 67 proteins were classified as stable interactors of the proteasome complex, whereas 16 of them were identified as dynamic interactors. The methods reported here provide a valuable expansion of proteomics technologies for identification of important but previously unidentifiable interacting proteins. Molecular & Cellular Proteomics 7:46 -57, 2008.Protein-protein interactions are one of the major mechanisms for controlling protein functions in various cellular processes. To fully understand these functions, global mapping of protein-protein interactions has become a major goal in current proteomics research. MS in combination with affinity purification in particular has evolved as a powerful tool for deciphering protein interaction networks at the proteome level (1-6). Although conventional affinity purification can preserve stable interactions under native conditions, we have shown recently that affinity purification coupled with in vivo crosslinking can extend identification of interacting proteins by capturing weak affinity or transient interactors (7). In contrast to stably interacting proteins, dynamically interacting proteins associate and dissociate with their binding partners at high on/off rates and are likely to be more susceptible to rapid regulation in response to cellular cues. Their identification is of great significance to proteomics research but is considerably challenging as efficient strategies to distinguish between stable and dynamic components of protein complexes are currently lacking. A new development that adds the ability to describe protein complex dynamics is thus needed for the advancement of interactive proteomics.Identification of specific protein interactions has been successfully achieved by quantitative MS using stable isotope labeling such as the stable isotope labeling of amino acids in cell culture (SILAC) 1 strategy, which allows effective discrimination ag...

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Mass Spectrometric Characterization of the Affinity-Purified Human 26S Proteasome Complex

Cited by 248 publications

References 43 publications

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Reference Measurement Procedure Development for C-Reactive Protein in Human Serum

Structural basis for dynamic regulation of the human 26S proteasome

Identifying Dynamic Interactors of Protein Complexes by Quantitative Mass Spectrometry

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