HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification.

Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol. HILAQ was successfully applied to the HT22 oxytosis model. 226 proteins were found to have a two-fold change in abundance, and 108 proteins were enriched in the cell death pathway, demonstrating the utility of HT22 cells as a tool to study the molecular details of cell death involved in neurodegenerative diseases. The HILAQ strategy simplifies the analysis of newly synthesized proteomes through the use of isobaric labels and achieves higher sensitivity than previously published methods.

Quantitative analysis of newly synthesized proteins.

Measuring proteome response to perturbations is critical for understanding the underlying mechanisms involved. Traditional quantitative proteomic methods are limited by the large numbers of proteins in the proteome and the mass spectrometer's dynamic range. A previous method uses the biorthogonal reagent azidohomoalanine (AHA), a methionine analog, for labeling, enrichment and detection of newly synthesized proteins (NSPs). Newly synthesized AHA proteins can be coupled to biotin via CuAAC-mediated click chemistry and enriched using avidin-based affinity purification. The combination of AHA-mediated NSP labeling with metabolic stable isotope labeling allows quantitation of low-abundant, newly secreted proteins by mass spectrometry (MS). However, the resulting multiplicity of labeling complicates NSP analysis. We developed a new NSP quantification strategy, called HILAQ (heavy isotope-labeled azidohomoalanine quantification), that uses a heavy isotope-labeled AHA molecule to enable NSP labeling, enrichment, identification and quantification. In addition, the AHA-peptide enrichment used in HILAQ improves both the identification and quantification of NSPs over AHA-protein enrichment. Here, we provide a description of the HILAQ method that includes procedures for (i) pulse-labeling and harvesting NSPs; (ii) addition of biotin by click reaction; (iii) protein precipitation; (iv) protein digestion; (v) enrichment of AHA-biotin peptides by NeutrAvidin beads and four-step elution; (vi) MS analysis; and (vii) data analysis for the identification and quantification of NSPs by ProLuCID and pQuant. We demonstrate our HILAQ approach by identifying NSPs from cell cultures, but we anticipate that it can be adapted for applications in animal models. The whole protocol takes ~6 d to complete.

Inhibition of the Staphylococcus aureus sortase transpeptidase SrtA by phosphinic peptidomimetics.

During pathogenesis, Gram-positive bacteria utilize surface protein virulence factors such as the MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) to aid the initiation and propagation of infection through adherence to host endothelial tissue and immune system evasion. These virulence-associated proteins generally contain a C-terminal LPXTG motif that becomes covalently anchored to the peptidoglycan biosynthesis intermediate lipid II. In Staphylococcus aureus, deletion of the sortase isoform SrtA results in marked reduction in virulence and infection potential, making it an important antivirulence target. Here we describe the chemical synthesis and kinetic characterization of a nonhydrolyzable phosphinic peptidomimetic inhibitor of SrtA derived from the LPXTG substrate sequence.

Chemistry and biology of the ramoplanin family of peptide antibiotics.

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.