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1.  Towards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging 
Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both fundamental studies and lipid marker discovery. In this paper, we report the identification and the localization of specific isobaric minor phospholipids in human breast cancer xenografts by FTICR MALDI imaging supported by histochemistry. These potential candidates can be further confirmed by liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) after extraction from the region of interest defined by MALDI imaging. Finally, this study highlights the importance of characterizing the heterogeneous distribution of low-abundant lipid species, relevant in complex histological samples for biological purposes.
doi:10.3390/ijms141224560
PMCID: PMC3876128  PMID: 24351834
MALDI mass spectrometry imaging; FTICR mass spectrometry; lipidomics; hypoxia; necrosis; cancer
2.  Proteomic Investigation of Aphid Honeydew Reveals an Unexpected Diversity of Proteins 
PLoS ONE  2013;8(9):e74656.
Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective.
doi:10.1371/journal.pone.0074656
PMCID: PMC3783439  PMID: 24086359
3.  The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction 
PLoS ONE  2013;8(9):e68014.
The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.
doi:10.1371/journal.pone.0068014
PMCID: PMC3779199  PMID: 24073192
4.  Identification of stromal proteins overexpressed in nodular sclerosis Hodgkin lymphoma 
Proteome Science  2011;9:63.
Hodgkin lymphoma (HL) represents a category of lymphoid neoplasms with unique features, notably the usual scarcity of tumour cells in involved tissues. The most common subtype of classical HL, nodular sclerosis HL, characteristically comprises abundant fibrous tissue stroma. Little information is available about the protein composition of the stromal environment from HL. Moreover, the identification of valid protein targets, specifically and abundantly expressed in HL, would be of utmost importance for targeted therapies and imaging, yet the biomarkers must necessarily be accessible from the bloodstream. To characterize HL stroma and to identify potentially accessible proteins, we used a chemical proteomic approach, consisting in the labelling of accessible proteins and their subsequent purification and identification by mass spectrometry. We performed an analysis of potentially accessible proteins in lymph node biopsies from HL and reactive lymphoid tissues, and in total, more than 1400 proteins were identified in 7 samples. We have identified several extracellular matrix proteins overexpressed in HL, such as versican, fibulin-1, periostin, and other proteins such as S100-A8. These proteins were validated by immunohistochemistry on a larger series of biopsy samples, and bear the potential to become targets for antibody-based anti-cancer therapies.
doi:10.1186/1477-5956-9-63
PMCID: PMC3200160  PMID: 21975223
Biomarker discovery; Lymphoma; Mass spectrometry; Tumour targeting
5.  d(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads 
Nucleic Acids Research  2011;39(17):7848-7857.
Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K+ or NH4+ ions, through the 5′-5′ stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads.
doi:10.1093/nar/gkr489
PMCID: PMC3177218  PMID: 21715378
6.  Cation Involvement in Telomestatin Binding to G-Quadruplex DNA 
Journal of Nucleic Acids  2010;2010:121259.
The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show the incorporation of one extra ammonium ion in the telomestatin complexes. Experiments on telomestatin alone also show that the telomestatin alone is able to coordinate cations in a similar way as a crown ether. Finally, density functional theory calculations suggest that in the G-quadruplex-telomestatin complex, potassium or ammonium cations are located between the telomestatin and a G-quartet. This study underlines that monovalent cation coordination capabilities should be integrated in the rational design of G-quadruplex binding ligands.
doi:10.4061/2010/121259
PMCID: PMC2911584  PMID: 20700418
7.  Tetramolecular G-quadruplex formation pathways studied by electrospray mass spectrometry 
Nucleic Acids Research  2010;38(15):5217-5225.
Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG5T, in ammonium acetate. The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified. The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant. However, these tetramers do not have their four strands perfectly aligned to give five G-quartets: the structures contain one ammonium ion too few, and ion mobility spectrometry shows that their conformation is more extended. (iv) At 4°C, the rearrangement of the kinetically trapped tetramers with presumably slipped strand(s) into the perfect G-quadruplex structure is extremely slow (not complete after 4 months). We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly.
doi:10.1093/nar/gkq208
PMCID: PMC2926595  PMID: 20400500
8.  Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines 
Introduction
Activator protein-2 (AP-2) α and AP-2γ transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity.
Methods
Ku proteins were identified as AP-2α interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression.
Results
Nuclear proteins from BT-474 cells overexpressing AP-2α and AP-2γ were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2α activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2α on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2α and AP-2γ siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2α and AP-2γ or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2α and AP-2γ. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter.
Conclusions
Ku proteins in interaction with AP-2 (α and γ) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.
doi:10.1186/bcr2450
PMCID: PMC2815545  PMID: 19906305
9.  Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes 
BMC Genomics  2009;10:362.
Background
Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro.
Results
We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q.
Conclusion
This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.
doi:10.1186/1471-2164-10-362
PMCID: PMC2736202  PMID: 19660104
10.  Cell Membrane Proteomic Analysis Identifies Proteins Differentially Expressed in Osteotropic Human Breast Cancer Cells12 
Neoplasia (New York, N.Y.)  2008;10(9):1014-1020.
Metastatic breast cancer cells are characterized by their high propensity to colonize the skeleton and form bone metastases, causing major morbidity and mortality. Identifying key proteins involved in the osteotropic phenotype would represent a major step toward the development of both new prognostic markers and new effective therapies. Cell surface proteins differentially expressed in cancer cells are preferred potential targets for antibody-based targeted therapies. In this study, using cell surface biotinylation and a mass spectrometric approach, we have compared the profile of accessible cell surface proteins between the human breast cancer cell line MDA-MB-231 and its highly osteotropic B02 subclone. This strategy allowed the identification of several proteins either up- or downregulated in the osteotropic cell line, and differential protein expressions were validated using antibody-based techniques. Class I HLAs were down-regulated in the bone metastatic variant, whereas αvβ3 integrins, among others, were consistently up-regulated in this latter cell line. These results show that comprehensive profiling of the cell surface proteome of mother cancerous cell lines and derived organ-specific metastatic cell lines provides an effective approach for the identification of potential accessible marker proteins for both prognosis and antibody-based targeted therapies.
PMCID: PMC2517647  PMID: 18714363
11.  Determination of affinity, stoichiometry and sequence selectivity of minor groove binder complexes with double-stranded oligodeoxynucleotides by electrospray ionization mass spectrometry 
Nucleic Acids Research  2002;30(16):e82.
Electrospray mass spectrometry was evaluated regarding the reliability of the determination of the stoichiometries and equilibrium association constants from single spectra. Complexes between minor groove binders (Hoechst 33258, Hoechst 33342, DAPI, netropsin and berenil) and 12mer oligonucleotide duplexes with a central sequence (A/T)4 flanked by G/C base pairs were chosen as model systems. To validate the electrospray ionization mass spectrometry (ESI-MS) method, comparisons were made with circular dichroism and fluorescence spectroscopy measurements. ESI-MS allowed the detection of minor (2 drug + DNA) species for Hoechst 33258, Hoechst 33342, DAPI and berenil with duplex d(GGGG(A/T)4GGGG)· d(CCCC(A/T)4CCCC), which were undetectable with the other techniques. Assuming that the duplexes and the complexes have the same electrospray response factors, the equilbrium association constants of the 1:1 and 2:1 complexes were determined by ESI-MS, and the values show a good quantitative agreement with fluorescence determined constants for Hoechst 33258 and Hoechst 33342. It is also shown that ESI-MS can quickly give reliable information on the A/T sequence selectivity of a drug: the signal of a complex is directly related to the affinity of the drug for that particular duplex. The potential of ESI-MS as a qualitative and quantitative affinity screening method is emphasized.
PMCID: PMC134255  PMID: 12177310
12.  Biochemical Characterization of the Pseudomonas aeruginosa 101/1477 Metallo-β-Lactamase IMP-1 Produced by Escherichia coli 
The blaIMP gene coding for the IMP-1 metallo-β-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21(DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 metallo-β-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-β-lactamase was not pH dependent.
PMCID: PMC89223  PMID: 10103197
13.  Mercury immune toxicity in harbour seals: links to in vitro toxicity 
Environmental Health  2008;7:52.
Background
Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes).
Methods
Total mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 μM). The expression of cytokines (IL-2, IL-4 and TGF-β) was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 μM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 μM (n = 3).
Results
The results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 μM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-β mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 μM). Proteomics showed some variation in the protein expression profile (e.g. vimentin).
Conclusion
Our results suggest that seal and human PBMCs react in a comparable way to MeHg in vitro exposure with, however, larger inter-individual variations. MeHg could be an additional cofactor in the immunosuppressive pollutant cocktail generally described in the blood of seals and this therefore raises the possibility of additional additive effects in the marine mammal immune system.
doi:10.1186/1476-069X-7-52
PMCID: PMC2600635  PMID: 18959786
14.  Proteome alteration induced by hTERT transfection of human fibroblast cells 
Proteome Science  2008;6:12.
Background
Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation.
Results
2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis.
Conclusion
We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions.
doi:10.1186/1477-5956-6-12
PMCID: PMC2386453  PMID: 18419814

Results 1-14 (14)