Proteomics Evaluation of Chemically Cleavable Activity-based Probes

Fonović, Marko; Verhelst, Steven H. L.; Sorum, Mark T.; Bogyo, Matthew

doi:10.1074/mcp.m700124-mcp200

Cited by 82 publications

(55 citation statements)

References 27 publications

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“…For limited trypsin digestion of bacterium-bound proteins, a modification of the on-bead digestion method was employed (31). Bacterial pellets (10 9 bacteria) carrying bound salivary proteins were washed three times with 50 mM ammonium bicarbonate buffer, pH 8 (1 ml).…”

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confidence: 99%

Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus

et al. 2013

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bProteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1 gp-340 , mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen. Saliva plays a key role in host defense against invading pathogens (1-4). Among the more than 2,000 proteins and peptides found in saliva (5), many exhibit direct antimicrobial activity (6). Others can bind to bacteria to facilitate either their colonization on oral surfaces or their clearance from the oral cavity through agglutination (7,8). It has been suggested that systemic pathogens can be killed, inactivated, or agglutinated by salivary components and, thus, become cleared from the oral cavity through swallowing, thereby preventing them from colonizing the oral cavity of healthy individuals (2, 9). Thus, binding of salivary proteins to pathogens is thought to play an important role in preventing systemic infections. In hospitalized patients, the protective and antimicrobial functions of saliva, which play a crucial role in host defense against invading pathogens (1-3), are frequently impaired by reduction of salivary flow or lack of salivary secretion (9-11). Under such conditions of dry mouth and poor oral hygiene, the normal commensal oral microflora shifts to a community that harbors a higher number of pathogens (12, 13).Among the various systemic pathogens in the oral cavity, attention has been given to Staphylococcus aureus (14, 15), since both endocarditis and pneumonia have been related to oral colonization by this organism (16,17). Studies have shown the occurrence of S. aureus in oral biofilm and saliva of healthy individuals (18), bu...

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confidence: 99%

Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus

et al. 2013

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“…Moreover, the site of modification can confirm the mechanism of probe incorporation. Tryptic peptides containing the active site of cathepsins were not found in our current experiments, as these are notoriously difficult to identify because of their large size (16).…”

Section: Discussionmentioning

confidence: 75%

“…The commercially available disulfide linker can be cleaved under mild conditions, but it suffers from premature cleavage in reducing media such as the intracellular environment and reducing buffers used for click chemistry and in vitro reactions of cysteine proteases. Therefore, a variety of alternative linkers for proteomics applications have been reported, including a sterically hindered disulfide (14), diazobenzenes (15)(16)(17)(18)(19), hydrazones (20,21), silanes (22), light sensitive linkers (23)(24)(25), tobacco etch virus protease sensitive linkers (26,27), and a levulinoyl-based linker (28). The synthesis of some of these linkers is lengthy or difficult to scale up, which limits their general application in chemical proteomics.…”

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confidence: 99%

A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

Yang

Hahne

Küster

et al. 2013

Molecular & Cellular Proteomics

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The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion. Molecular & Cellular

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“…Addendum-After submission of this work, two studies describing cleavable probes for functional proteomics were published (30,31). * This work was supported by National Institutes of Health Grants CA087660 (to B. F. C.), CA37393 (to B. R.…”

Section: Figmentioning

confidence: 99%