High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

Berridge, G.; Chalk, R.; D’Avanzo, Nazzareno; Doyle, D.A.; Kim, Jung-In; Xia, Xiaobing; Burgess-Brown, N.; deRiso, Antonio; Carpenter, E.P.; Gileadi, O.

doi:10.1016/j.ab.2010.11.008

Cited by 22 publications

(26 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although the H-DivIB/DivIC/FtsL complex was successfully purified in several detergents, DDM was selected for larger scale experiments due to its compatibility with mass spectrometry measurements [46]. …”

Section: Resultsmentioning

confidence: 99%

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

et al. 2013

View full text Add to dashboard Cite

The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

show abstract

Section: Resultsmentioning

confidence: 99%

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Gel bands corresponding to stable domains were excised from the gel and overlaid with 10% MeOH at 4 °C. In-gel tryptic digests of each gel slice were performed as described previously 37 . A Dionex U3000 nano-HPLC coupled to a Bruker Esquire HCT ion trap mass spectrometer was used to perform LC–MS/MS 37 .…”

Section: Methodsmentioning

confidence: 99%

“…In-gel tryptic digests of each gel slice were performed as described previously 37 . A Dionex U3000 nano-HPLC coupled to a Bruker Esquire HCT ion trap mass spectrometer was used to perform LC–MS/MS 37 . Samples were also removed from the original tryptic digest reaction for intact mass analysis at the same time points.…”

Section: Methodsmentioning

confidence: 99%

The PHD and Chromo Domains Regulate the ATPase Activity of the Human Chromatin Remodeler CHD4

Watson

Mahajan

Mertens

et al. 2012

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.

show abstract

“…Since lipids and MSP are present in Nanodiscs at least at a 65:2:1 lipid/MSP/membrane protein ratio, membrane proteins in Nanodiscs are in relatively low abundance and are in the presence of high lipid concentrations. There are several sample preparation methods that have been developed to improve detection of membrane proteins in detergent and that could potentially be applied to Nanodisc samples [15,23]. However, sample preparation that involves chromatography is generally more time and material intensive.…”

Section: Introductionmentioning

confidence: 99%

Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs

2011

View full text Add to dashboard Cite

Nanodiscs have become a leading technology to solubilize membrane proteins for biophysical, enzymatic, and structural investigations. Nanodiscs are nanoscale, discoidal lipid bilayers surrounded by an amphipathic membrane scaffold protein (MSP) belt. A variety of analytical tools has been applied to membrane proteins in Nanodiscs, including several recent mass spectrometry studies. Mass spectrometry of full-length proteins is an important technique for analyzing protein modifications, for structural studies, and for identification of proteins present in binding assays. However, traditional MALDI-TOF mass spectrometry methods for analyzing full-length membrane proteins solubilized in Nanodiscs are limited by strong signal from the MSP belt and weak signal from the membrane protein inside the Nanodisc. Herein we show that an optimized ultra-thin layer MALDI sample preparation technique dramatically enhances the membrane protein signal and nearly completely eliminates the MSP signal. First shot MALDI and MALDI imaging are used to characterize the spots formed by the ultra-thin layer method. Furthermore, the membrane protein enhancement and MSP suppression is shown to be independent of the type of membrane protein and is applicable to mixtures of membrane proteins in Nanodiscs.

show abstract

High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

Cited by 22 publications

References 22 publications

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

The PHD and Chromo Domains Regulate the ATPase Activity of the Human Chromatin Remodeler CHD4

Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs

Contact Info

Product

Resources

About