Mass Spectrometric Analysis of Cyanogen Bromide Fragments of Integral Membrane Proteins at the Picomole Level: Application to Rhodopsin

Kraft, Paul; Mills, John; Dratz, Edward A.

doi:10.1006/abio.2001.5072

Cited by 36 publications

(34 citation statements)

References 38 publications

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“…Due to the relative lack of Lys/Arg residues, TM ␣-helices are typically present as relatively large, hydrophobic peptides in tryptic digest samples and have been shown to present difficulties for mass analysis (48,49). In the present study, the proposed gp91 phox TM domains 2 and 6 were readily detected by both MALDI and LC-MS/MS, and the detection of these fragments was undoubtedly facilitated by the presence of trypsin cleavage sites within the membrane-spanning region.…”

Section: Discussionmentioning

confidence: 99%

“…phox -Despite the fact that mass spectrometry represents a powerful technique for protein structural characterization, integral membrane proteins can present unique difficulties (48,49), and these methods have not previously been applied for the mass analysis of Cyt b. In the present study, Cyt b was immunoaffinity-purified from human neutrophil membrane fractions and resolved by SDS-PAGE to separate the gp91 phox and p22 phox subunits for in-gel tryptic digestion (Fig.…”

Section: Mass Analysis Of Human Neutrophil Gp91mentioning

confidence: 99%

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Analysis of Human Phagocyte Flavocytochrome b558 by Mass Spectrometry

Taylor

Baniulis

Burritt

et al. 2006

Journal of Biological Chemistry

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The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91 phox subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22 phox subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91 phox subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91 phox subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.

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Section: Discussionmentioning

confidence: 99%

Section: Mass Analysis Of Human Neutrophil Gp91mentioning

confidence: 99%

Analysis of Human Phagocyte Flavocytochrome b558 by Mass Spectrometry

Taylor

Baniulis

Burritt

et al. 2006

Journal of Biological Chemistry

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“…The eluted samples containing Ste2p (ϳ20 g) were dried by vacuum centrifugation (Thermo Scientific, Waltham, MA) and then dissolved in 100% TFA containing 10 mg/ml CNBr. Deionized distilled water was then added to adjust the final TFA concentration to 80%, and the samples were incubated at 37°C in the dark for 18 h (18,27). The samples were dried by vacuum centrifugation and washed three times with deionized distilled water, and then 1 M Tris (pH 8.0) was added to neutralize the acidic mixture.…”

Section: Digestion Of Cross-linked Ste2pmentioning

confidence: 99%

Identification of Residue-to-residue Contact between a Peptide Ligand and Its G Protein-coupled Receptor Using Periodate-mediated Dihydroxyphenylalanine Cross-linking and Mass Spectrometry

Umanah

Huang

Ding

et al. 2010

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs)4 comprise the largest family of cell surface proteins present in the human genome responsible for communication between the internal and external environments in response to signal molecules, including hormones, pheromones, and neurotransmitters. GPCRs are characterized by the presence of seven membrane-spanning helical segments separated by alternating intracellular and extracellular loop regions (1-3). Despite their diverse ligands, all GPCRs perform similar functions, coupling the binding of ligands to the activation of specific heterotrimeric guanine nucleotide-binding proteins (G proteins), leading to the modulation of downstream effector proteins and molecules. Due to their involvement in a multitude of physiological functions, GPCRs are targeted by ϳ50% of the human drugs currently marketed (1,4,5). Detailed information about GPCR-ligand interactions is critical for our understanding of GPCR-ligand binding and function.Despite the differences in the binding mechanisms of various ligand groups in the GPCR family, there are some similarities that span this superfamily of proteins (1, 3, 6 -8). Ligand binding triggers conformational changes at the extracellular and transmembrane regions of the receptor, which are then propagated to the cytoplasmic surfaces, leading to G protein coupling and activation. Thus, ligand binding leads to the alteration of existing interhelical interactions, thereby promoting a set of new interactions that leads to a energetically favorable activated conformational state of the receptor (8, 9). Large ligands, such as proteins, bind to the extracellular loops of GPCRs, whereas small molecules like adrenergic agents bind within the transmembrane region of the receptor. Within the peptide-binding GPCRs, a combination of ligand binding to the extracellular loops followed by ligand penetra-

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“…Ball et al [15] demonstrated complete mapping of BR and rhodopsin with CNBr, but their method used a large quantity of peptides (,250 mg) compared to the method reported here, which utilizes only 4.8 mg of total peptides per LC run. Kraft et al [14] also obtained complete peptide mapping of integral membrane rhodopsin using CNBr with MALDI-MS for detection. Further, 99% coverage was obtained by Ablonczy et al [26] using CNBr, but this method required protein precipitation, which often leads to protein loss when preparing complex mixtures.…”

Section: Resultsmentioning

confidence: 99%

“…Being readily available and previously well characterized, bacteriorhodopsin (BR) [14][15][16][17][18][19][20] was chosen as the model to develop and demonstrate improved methods for characterizing integral membrane proteins. The seven a-helical transmembrane regions of BR are of particular interest in this model system since detection of these regions would likely be also applicable to complex cellular membrane protein mixtures for application in high-throughput proteomics.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of enzymatic digestion and liquid chromatography‐mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin

Hixson

Rodríguez²,

Camp

et al. 2002

Electrophoresis

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A method for the complete peptide mapping of the model integral membrane protein bacteri-orhodopsin is demonstrated. Utilizing more effective enzymatic digestion, procedures with capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and tandem mass spectrometry (MS/MS), all predicted tryptic digestion products were detected, as well as peptides from all previously reported post-translational modifications of bacteriorhodopsin. A significant contribution of chymotryptic-like digestion products was also observed. A characterization of the behavior of hydrophobic integral membrane peptides in a reversed-phase liquid chromatographic separation is also provided. The method reported here offers improved compatibility of the solubilizing reagents with both the chromatography and mass spectrometry, rendering it suitable for high-throughput proteomic applications.

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Mass Spectrometric Analysis of Cyanogen Bromide Fragments of Integral Membrane Proteins at the Picomole Level: Application to Rhodopsin

Cited by 36 publications

References 38 publications

Analysis of Human Phagocyte Flavocytochrome b558 by Mass Spectrometry

Analysis of Human Phagocyte Flavocytochrome b558 by Mass Spectrometry

Identification of Residue-to-residue Contact between a Peptide Ligand and Its G Protein-coupled Receptor Using Periodate-mediated Dihydroxyphenylalanine Cross-linking and Mass Spectrometry

Evaluation of enzymatic digestion and liquid chromatography‐mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin

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