André M. N. Silva

Post-translational modifications and mass spectrometry detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.Lyden and colleagues use asymmetric flow field-flow fractionation to classify nanoparticles derived from cell lines and human samples, including previously uncharacterized large, Exo-L and small, Exo-S, exosome subsets.

Iron(III) Fluorinated Porphyrins: Greener Chemistry from Synthesis to Oxidative Catalysis Reactions

Iron(III) fluorinated porphyrins play a central role in the biomimetics of heme enzymes and enable cleaner routes to the oxidation of organic compounds. The present work reports significant improvements in the eco-compatibility of the synthesis of 5,10,15,20-tetrakis-pentafluorophenylporphyrin (H2TPFPP) and the corresponding iron complex [Fe(TPFPP)Cl], and the use of [Fe(TPFPP)Cl] as an oxidation catalyst in green conditions. The preparations of H2TPFPP and [Fe(TPFPP)Cl] typically use toxic solvents and can be made significantly greener and simpler using microwave heating and optimization of the reaction conditions. In the optimized procedure it was possible to eliminate nitrobenzene from the porphyrin synthesis and replace DMF by acetonitrile in the metalation reaction, concomitant with a significant reduction of reaction time and simplification of the purification procedure. The Fe(III)porphyrin is then tested as catalyst in the selective oxidation of aromatics at room temperature using a green oxidant (hydrogen peroxide) and green solvent (ethanol). Efficient epoxidation of indene and selective oxidation of 3,5-dimethylphenol and naphthalene to the corresponding quinones is observed.

Chlorogenic acid–arabinose hybrid domains in coffee melanoidins: Evidences from a model system

Arabinose from arabinogalactan side chains was hypothesized as a possible binding site for chlorogenic acids in coffee melanoidins. To investigate this hypothesis, a mixture of 5-O-caffeoylquinic acid (5-CQA), the most abundant chlorogenic acid in green coffee beans, and (α1 → 5)-L-arabinotriose, structurally related to arabinogalactan side chains, was submitted to dry thermal treatments. The compounds formed during thermal processing were identified by electrospray ionization mass spectrometry (ESI-MS) and characterized by tandem MS (ESI-MS(n)). Compounds composed by one or two CQAs covalently linked with pentose (Pent) residues (1-12) were identified, along with compounds bearing a sugar moiety but composed exclusively by the quinic or caffeic acid moiety of CQAs. The presence of isomers was demonstrated by liquid chromatography online coupled to ESI-MS and ESI-MS(n). Pent1-2CQA were identified in coffee samples. These results give evidence for a diversity of chlorogenic acid-arabinose hybrids formed during roasting, opening new perspectives for their identification in melanoidin structures.

Targeted O‐glycoproteomics explored increased sialylation and identified MUC16 as a poor prognosis biomarker in advanced‐stage bladder tumours

Bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation on the cell surface, which may be explored for improving disease management. In a search for prognosis biomarkers and novel therapeutic targets we have screened, using immunohistochemistry, a series of bladder tumours with differing clinicopathology for short‐chain O‐glycans commonly found in glycoproteins of human solid tumours. These included the Tn and T antigens and their sialylated counterparts sialyl‐Tn(STn) and sialyl‐T(ST), which are generally associated with poor prognosis. We have also explored the nature of T antigen sialylation, namely the sialyl‐3‐T(S3T) and sialyl‐6‐T(S6T) sialoforms, based on combinations of enzymatic treatments. We observed a predominance of sialoglycans over neutral glycoforms (Tn and T antigens) in bladder tumours. In particular, the STn antigen was associated with high‐grade disease and muscle invasion, in accordance with our previous observations. The S3T and S6T antigens were detected for the first time in bladder tumours, but not in healthy urothelia, highlighting their cancer‐specific nature. These glycans were also overexpressed in advanced lesions, especially in cases showing muscle invasion. Glycoproteomic analyses of advanced bladder tumours based on enzymatic treatments, Vicia villosa lectin‐affinity chromatography enrichment and nanoLC‐ESI‐MS/MS analysis resulted in the identification of several key cancer‐associated glycoproteins (MUC16, CD44, integrins) carrying altered glycosylation. Of particular interest were MUC16 STn+‐glycoforms, characteristic of ovarian cancers, which were found in a subset of advanced‐stage bladder tumours facing the worst prognosis. In summary, significant alterations in the O‐glycome and O‐glycoproteome of bladder tumours hold promise for the development of novel noninvasive diagnostic tools and targeted therapeutics. Furthermore, abnormal MUC16 glycoforms hold potential as surrogate biomarkers of poor prognosis and unique molecular signatures for designing highly specific targeted therapeutics.

Cross‐oxidation of angiotensin II by glycerophosphatidylcholine oxidation products

Peptide and protein lipoxidation is a deleterious process which has been related to several degenerative conditions. In the present study, the interaction of lipid secondary oxidation products with peptides was investigated by evaluating the modifications occurring to angiotensin II (Ang-II) in the presence of an oxidizing polyunsaturated glycerophospholipid (1-palmitoyl-2-arachidonoyl-glycerophosphatidylcholine, PAPC). PAPC oxidation was promoted by Fenton chemistry and the oxidation products were incubated with Ang-II. The reaction products were finally analysed by off-line nanospray high-performance liquid chromatography/matrix-assisted laser desorption/ionization tandem mass spectrometry (nano-HPLC/MALDI-TOF-MS/MS). Ang-II was found to form adducts with 26 different aldehydes, leading to 37 distinct reaction products. Modification of Ang-II occurred through reaction with reactive carbonyl species (RCS) originating from fatty acyl chain cleavage, while interactions with the oxidized phospholipid could not be detected. Adduction was observed to occur both by Michael and Schiff base mechanisms, most prevalently taking place at the peptide N-terminus or the arginine residue. Histidine modification could only be demonstrated to occur via Michael addition with two aldehydes: 4-hydroxy-2-nonenal (HNE) and 2-octenal. The highly reactive 4-oxo-2-nonenal (ONE) was shown to react preferentially with the arginine side chain, while malondialdehyde addition could only be confirmed at the N-terminus. Aspartic acid oxidative decarboxylation, amino acid side chain oxidation, multiple adduction or peptide cross-links could not be perceived. The inability to detect these reaction products is indicative of their low abundance or non-existence in competitive reaction conditions. The multiplicity of peptide modifications described emphasizes the complexity of lipoxidation, the effects of which are not possible to fully understand by the evaluation of independent reaction products.

Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O -glycosylation extension

Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may offer potential to treat tumour hypoxic niches harbouring more malignant cells.

Characterization of in vitro protein oxidation using mass spectrometry: a time course study of oxidized alpha-amylase.

The study of protein damage by oxidative processes and its influence on protein activity is central to understanding the deleterious effects of oxidative stress on biological systems. This paper will focus on the study of enzyme inactivation by oxidative modifications, utilizing α-amylase from Bacillus species. (BAA) as a model protein. Oxidative stress was induced using metal catalyzed oxidation (MCO). The enzymatic activity of BAA was correlated with the oxidative damage induced to the protein. Off-line nano-HPLC-MALDI-TOF/TOF-MS was used to characterize the oxidative modifications occurring to the protein. Additionally, semi-quantitative analysis was employed in order to evaluate the significance of the various oxidative modifications. BAA oxidation was found to be deleterious to its enzymatic activity. A total of 10 amino acid residues were found to have an oxidation degree above 50%, out of which eight were methionine and tryptophan. Residues in the proximity of key structural elements were found to be particularly susceptible to oxidation. The oxidative process was found to be governed by the nature of the amino acid residues side chain and, to a lesser extent, their location within the three dimensional structure of the protein.

In silico approaches for unveiling novel glycobiomarkers in cancer

Glycosylation is one of the most common and dynamic post-translational modification of cell surface and secreted proteins. Cancer cells display unique glycosylation patterns that decisively contribute to drive oncogenic behavior, including disease progression and dissemination. Moreover, alterations in glycosylation are often responsible for the creation of protein signatures holding significant biomarker value and potential for targeted therapeutics. Accordingly, many analytical protocols have been outlined for the identification of abnormally glycosylated proteins by mass spectrometry. Nevertheless, very few studies undergo a comprehensive mining of the generated data. Herein, we build on bladder cancer O-glycoproteomics datasets resulting from a hyphenated technique comprising enrichment by Vicia villosa agglutinin (VVA) lectin and nanoLC-ESI-MS/MS to propose an in silico step-by-step tutorial (Panther, UniProtKB, NetOGlyc, NetNGlyc, Oncomine, Cytoscape) for biomarker discovery in cancer. We envisage that this approach may be generalized to other mass spectrometry-based analytical approaches, including N-glycoproteomics studies, and different types of cancers. SIGNIFICANCE The glycoproteome is an important source of cancer biomarkers holding tremendous potential for targeted therapeutics. We now present an in silico roadmap for comprehensive interpretation of big data generated by mass spectrometry-based glycoproteomics envisaging the identification of clinically relevant glycobiomarkers.

1,3-Dipolar cycloadditions with meso-tetraarylchlorins – site selectivity and mixed bisadducts

The 1,3-dipolar cycloaddition reaction of meso-tetraarylporphyrins with nitrones gives isoxazolidine-fused chlorins. Depending on the substitution pattern on the meso-aryl groups and the nitrone, the chlorins can be obtained in high yields (up to 91%). Bacteriochlorin-type bisadducts are also obtained, although in low yield, from the reaction of meso-tetrakis(pentafluorophenyl)porphyrin with N-methyl, N-cyclohexyl or N-benzyl nitrone. The structure of a bis(N-benzyl isoxazolidine-fused) bacteriochlorin was determined by single-crystal X-ray diffraction and rationalized by DFT calculations. To further explore the nature of site selectivity in the formation of bisadducts, isomeric mixed bacteriochlorins and isobacteriochlorins were synthesized by two complementary routes that involved the sequential addition of two types of 1,3-dipoles to the porphyrin macrocycle: a nitrone and an azomethine ylide. The photophysical properties of the mixed bacteriochlorins were evaluated and the results were compared with the reported data for other meso-tetraarylbacteriochlorins.