Complementary Analysis of the Vegetative Membrane Proteome of the Human Pathogen Staphylococcus aureus

Wolff, Susanne; Hahne, Hannes; Hecker, Michael; Becher, Dörte

doi:10.1074/mcp.m700554-mcp200

Cited by 57 publications

(59 citation statements)

References 39 publications

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“…The enriched membrane protein fraction was prepared according to the workflow published by Eymann et al (78), omitting the n-dodecyl-β-D-maltoside treatment step. The preparation of the integral membrane peptides was carried out as described by Wolff et al (79). Sample preparation, mass spectrometric measurement, and subsequent data analysis were carried out as described by Otto et al (77).…”

Section: Methodsmentioning

confidence: 99%

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

Wenzel

Chiriac

Otto

et al. 2014

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

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Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH 2 . A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. mechanism of action | respiratory chain | hypoosmotic stress response | metallocenes

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Section: Methodsmentioning

confidence: 99%

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

Wenzel

Chiriac

Otto

et al. 2014

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

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303

329

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“…To further investigate a possible preference for hydrophobic peptides of either ionization technique, we additionally analyzed the membrane shaving fraction, purified and analyzed via LTQ-Orbitrap by Hessling et al, 2 with MALDI-TOF-TOF. The membrane shaving fraction especially contains the hydrophobic membrane-spanning domains of peptides (14) and had never been analyzed before with MALDI-MS. In the preparation of these fractions, cell membranes are spun down via ultracentrifugation and digested with Proteinase K to deplete the soluble loops of membraneassociated proteins.…”

Section: Biochemical Properties Of Preferentially Identified Peptides-mentioning

confidence: 99%

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Heßling

Büttner

Hecker

et al. 2013

Molecular & Cellular Proteomics

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Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LCelectrospray ionization workflows.Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis.Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF-and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments. One-dimensional gel-based liquid chromatography mass spectrometry (GeLC-MS) 1 is a well-established technique in life science. In combination with in vivo labeling approaches such as stable isotope labeling by amino acids in cell culture (SILAC) (1) or 15 N labeling (2), it allows the relative quantification of large numbers of proteins in a complex sample. Mass spectrometry (MS) measurements in such workflows are predominantly performed with electrospray ionization (ESI)-based mass spectrometers. Online coupling of a liquid chromatography (LC) system with fast MS spectra acquisition and high-mass-accuracy ESI instruments in conjunction with fractionation on both protein and peptide levels allows the analysis of very complex samples in a relatively short period of time (3). The identification and quantification of data can be done in an automatic or semi-automatic manner with a variety of well-established software packages (4).In proteomic research, matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) is mainly used for the analysis of non-complex protein samples that do not require fractionation on the peptide level by means of liquid chromatography. In particular, the analysis of single protein spots resulting from the twodimensional PAGE separation of complex samples is primarily carried out with this MS tec...

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“…As the cytoplasmic proteins of an organism can be identified with the routinely used procedures in the laboratories, most studies have recently focused at identification of membrane proteins (1)(2)(3)(4). The hydrophobic character of these latter molecules makes them poorly soluble in aqueous solvents and hence their separation by two-dimensional PAGE is very complicated (5)(6)(7)(8)(9)(10). Membrane proteins are invariably found to be associated with the cytoplasmic proteins even after using stringent preparation procedures (3,8,9).…”

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confidence: 99%

Identification of Outer Membrane Proteins from an Antarctic Bacterium Pseudomonas syringae Lz4W

Jagannadham

Abou-Eladab

Kulkarni

2011

Molecular & Cellular Proteomics

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Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on twodimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-timeof-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program.

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Complementary Analysis of the Vegetative Membrane Proteome of the Human Pathogen Staphylococcus aureus

Cited by 57 publications

References 39 publications

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Identification of Outer Membrane Proteins from an Antarctic Bacterium Pseudomonas syringae Lz4W

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