18O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

Introduction

Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.

Materials and Methods

In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent ¹⁸O/¹⁶O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to ¹⁸O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.

Conclusion

This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.     

Plasma Proteome Database as a resource for proteomics research

Plasma is one of the best studied compartments in the human body and serves as an ideal body fluid for the diagnosis of diseases. This report provides a detailed functional annotation of all the plasma proteins identified to date. In all, gene products encoded by 3778 distinct genes were annotated based on proteins previously published in the literature as plasma proteins and the identification of multiple peptides from proteins under HUPO's Plasma Proteome Project. Our analysis revealed that 51% of these genes encoded more than one protein isoform. All single nucleotide polymorphisms involving protein-coding regions were mapped onto the protein sequences. We found a number of examples of isoform-specific subcellular localization as well as tissue expression. This database is an attempt at comprehensive annotation of a complex subproteome and is available on the web at http://www.plasmaproteomedatabase.org.     

Effect of Red Bull energy drink on cardiovascular and renal function

Energy drink consumption has been anecdotally linked to the development of adverse cardiovascular effects in consumers, although clinical trials to support this link are lacking. The effects of Red Bull^® energy drink on cardiovascular and neurologic functions were examined in college-aged students enrolled at Winona State University. In a double-blind experiment where normal calorie and low calorie Red Bull^® were compared to normal and low calorie placebos, no changes in overall cardiovascular function nor blood glucose (mg/dL) were recorded in any participant (n = 68) throughout a 2-h test period. However, in the second experiment, nine male and twelve female participants subjected to a cold pressor test (CPT) before and after Red Bull^® consumption showed a significant increase in blood sugar levels pre- and post Red Bull^® consumption. There was a significant increase in diastolic blood pressure of the male volunteers immediately after submersion of the hand in the 5°C water for the CPT. Under the influence of Red Bull^®, the increase in diastolic pressure for the male participants during the CPT was negated. There were no significant changes in the blood pressure of the female participants for the CPT with or without Red Bull^®. Finally, the CPT was used to evaluate pain threshold and pain tolerance before and after Red Bull^® consumption. Red Bull^® consumption was associated with a significant increase in pain tolerance in all participants. These findings suggest that Red Bull^® consumption ameliorates changes in blood pressure during stressful experiences and increases the participants’ pain tolerance.     

The anandamide membrane transporter. Structure-activity relationships of anandamide and oleoylethanolamine analogs with phenyl rings in the polar head group region

A new series of anandamide and N-oleoylethanolamine analogs, most of which with aromatic moieties in the head group region, has been synthesized and evaluated as inhibitors of anandamide uptake. Some of them efficaciously inhibit the uptake process with K(i) values in the low micromolar range (2.4-21.2microM). Strict structural requisites are needed to observe a significant inhibition and in no case inhibition of fatty acid amidohydrolase overlaps with inhibition of anandamide uptake.     

Synthesis and biological evaluation of piperazinyl carbamates and ureas as fatty acid amide hydrolase (FAAH) and transient receptor potential (TRP) channel dual ligands

The evaluation of a series of piperazinyl carbamates and ureas, designed on the basis of previously reported TRPV1 antagonists and FAAH inhibitors, led to the identification of some ‘dual-action’ compounds targeting both FAAH and TRPV1 or TRPA1 receptors.     

Exploring the interest of 1,2-dithiolane ring system in peptide chemistry. Synthesis of a chemotactic tripeptide and x-ray crystal structure of a 4-amino-1,2-dithiolane-4-carboxylic acid derivative.

Due to their relevant biological functions and specific chemical reactivity 1,2-dithiolanes (five-membered cyclic disulfides) represent an emerging class of heterocyclic compounds. However, despite the extensive research centered on lipoic acid and its analogues, only very few data are at the present available on peptides containing this ring system. We report here synthesis, conformation and bioactivity of a fMLF-OMe analogue, namely For-Met-Adt-Phe-OMe (7), in which the residue of the 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) (4) replaces the central L-leucine. The crystal conformation of the synthetic intermediate Boc-Adt-OMe (5) is also described and compared to that of lipoic acid (R-1,2-dithiolane-3-pentanoic acid) (3) and asparagusic acid (1,2-dithiolane-4-carboxylic acid) (2).     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine-containing fMLF analogs.

In order to explore the properties of chemotactic N-formylpeptides containing isopeptide bonds within their backbones, a group of lysine-containing analogs of the prototypical chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO-Met or Boc-Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N alpha or N epsilon bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO-Met-Lys(Z)-Phe-OMe (2a, b), RCO-Met-Lys(Z-Phe)-OMe (3a, b), Z-Lys(RCO-Met)-Phe-OMe (4a, b), Z-Phe-Lys(RCO-Met)-OMe (5a, b), RCO-Met-Phe-Lys(Z)-OMe (6a, b) and Z-Lys(RCO-Met-Phe)-OMe (7a, b), with R=OC(CH3)(3 )and R=H for compounds a and b, respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b) and antagonists (compounds a). All N-formyl derivatives 2b-7b are less potent than fMLF-OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a-7a do not show antagonistic activity towards fMLF induced chemotaxis and O(2)(-) production, however, all these compounds except 4a antagonize lysozyme release by 60%.