Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information.

Affinity photo-cross-linking coupled to mass spectrometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ9), 18:1 (trans Δ9) PG, and DLoPG (18:2 cis Δ9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: C═C double bond formation on saturated fatty acids, covalent adducts formation via classical C-C bond, and Paternò-Büchi oxetane formation followed or not by fragmentation (retro-Paternò-Büchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction.

A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases.

Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol, we can monitor the methylation events over time on a peptide substrate. We detect in the same spectrum the modified and unmodified substrates, and the ratios of both signals are used to quantify the amount of methylated substrate. We first demonstrated the validity of the assay by determining inhibition parameters of two known inhibitors of the KMT SET7/9 ((R)-PFI-2 and sinefungin). Next, based on structural comparison with these inhibitors, we selected 42 compounds from a chemical library. We applied the MALDI-TOF assay to screen their activity as inhibitors of the KMT SET7/9. This study allowed us to determine inhibition constants as well as kinetic parameters of a series of SET7/9 inhibitors and to initiate a structure activity discussion with this family of compounds. This assay is versatile and can be easily adapted to other KMT substrates and enzymes as well as automatized.

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell-Penetrating Peptide Penetratin in Model Membranes.

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross-linked products by MALDI-TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.

Gender-Specific Toxicological Effects of Chronic Exposure to Pure Microcystin-LR or Complex Microcystis aeruginosa Extracts on Adult Medaka Fish.

Cyanobacterial blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to other organisms. However, their overall and specific implications for the health of aquatic organisms that are chronically and environmentally exposed to cyanobacteria producing hepatotoxins, such as microcystins (MCs), together with other bioactive compounds have still not been clearly established and remain difficult to assess. The medaka fish was chosen as the experimental aquatic model for studying the cellular and molecular toxicological effects on the liver after chronic exposures (28 days) to environmentally relevant concentrations of pure MC-LR, complex extracts of MC producing or nonproducing cyanobacterial biomasses, and of a Microcystis aeruginosa natural bloom. Our results showed a higher susceptibility of females to the different treatments compared to males at both the cellular and the molecular levels. Although hepatocyte lysis increased with MC-containing treatments, lysis always appeared more severe in the liver of females compare to males, and the glycogen cellular reserves also appeared to decrease more in the liver of females compared to those in the males. Proteomic investigations reveal divergent responses between males and females exposed to all treatments, especially for proteins involved in metabolic and homeostasis processes. Our observations also highlighted the dysregulation of proteins involved in oogenesis in female livers. These results suggest that fish populations exposed to cyanobacteria blooms may potentially face several ecotoxicological issues.

Phosphorylation of ABCB4 impacts its function: insights from disease-causing mutations.

UNLABELLED: The ABCB4 transporter mediates phosphatidylcholine (PC) secretion at the canalicular membrane of hepatocytes and its genetic defects cause biliary diseases. Whereas ABCB4 shares high sequence identity with the multidrug transporter, ABCB1, its N-terminal domain is poorly conserved, leading us to hypothesize a functional specificity of this domain. A database of ABCB4 genotyping in a large series of patients was screened for variations altering residues of the N-terminal domain. Identified variants were then expressed in cell models to investigate their biological consequences. Two missense variations, T34M and R47G, were identified in patients with low-phospholipid-associated cholelithiasis or intrahepatic cholestasis of pregnancy. The T34M and R47G mutated proteins showed no or minor defect, respectively, in maturation and targeting to the apical membrane, in polarized Madin-Darby Canine Kidney and HepG2 cells, whereas their stability was similar to that of wild-type (WT) ABCB4. By contrast, the PC secretion activity of both mutants was markedly decreased. In silico analysis indicated that the identified variants were likely to affect ABCB4 phosphorylation. Mass spectrometry analyses confirmed that the N-terminal domain of WT ABCB4 could undergo phosphorylation in vitro and revealed that the T34M and R47G mutations impaired such phosphorylation. ABCB4-mediated PC secretion was also increased by pharmacological activation of protein kinases A or C and decreased by inhibition of these kinases. Furthermore, secretion activity of the T34M and R47G mutants was less responsive than that of WT ABCB4 to protein kinase modulators. CONCLUSION: We identified disease-associated variants of ABCB4 involved in the phosphorylation of its N-terminal domain and leading to decreased PC secretion. Our results also indicate that ABCB4 activity is regulated by phosphorylation, in particular, of N-terminal residues.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.

A cationic motif upstream Engrailed2 homeodomain controls cell internalization through selective interaction with heparan sulfates.

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.

Muscle creatine kinase deficiency triggers both actin depolymerization and desmin disorganization by advanced glycation end products in dilated cardiomyopathy.

Alterations in the balance of cytoskeleton as well as energetic proteins are involved in the cardiac remodeling occurring in dilated cardiomyopathy (DCM). We used two-dimensional DIGE proteomics as a discovery approach to identify key molecular changes taking place in a temporally controlled model of DCM triggered by cardiomyocyte-specific serum response factor (SRF) knock-out in mice. We identified muscle creatine kinase (MCK) as the primary down-regulated protein followed by α-actin and α-tropomyosin down-regulation leading to a decrease of polymerized F-actin. The early response to these defects was an increase in the amount of desmin intermediate filaments and phosphorylation of the αB-crystallin chaperone. We found that αB-crystallin and desmin progressively lose their striated pattern and accumulate at the intercalated disk and the sarcolemma, respectively. We further show that desmin is a preferential target of advanced glycation end products (AGE) in mouse and human DCM. Inhibition of CK in cultured cardiomyocytes is sufficient to recapitulate both the actin depolymerization defect and the modification of desmin by AGE. Treatment with either cytochalasin D or glyoxal, a cellular AGE, indicated that both actin depolymerization and AGE contribute to desmin disorganization. Heat shock-induced phosphorylation of αB-crystallin provides a transient protection of desmin against glyoxal in a p38 MAPK-dependent manner. Our results show that the strong down-regulation of MCK activity contributes to F-actin instability and induces post-translational modification of αB-crystallin and desmin. Our results suggest that AGE may play an important role in DCM because they alter the organization of desmin filaments that normally support stress response and mitochondrial functions in cardiomyocytes.

MALDI-TOF mass spectrometry analysis of amphipol-trapped membrane proteins.

Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of Escherichia coli outer membrane protein A, a ß-barrel, eubacterial MP, (ii) Halobacterium salinarum bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga Chlamydomonas reinhardtii and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.

MALDI-TOF mass spectrometry: a powerful tool to study the internalization of cell-penetrating peptides.

This review summarizes the contribution of MALDI-TOF mass spectrometry in the study of cell-penetrating peptide (CPP) internalization in eukaryote cells. This technique was used to measure the efficiency of cell-penetrating peptide cellular uptake and cargo delivery and to analyze carrier and cargo intracellular degradation. The impact of thiol-containing membrane proteins on the internalization of CPP-cargo disulfide conjugates was also evaluated by combining MALDI-TOF MS with simple thiol-specific reactions. This highlighted the formation of cross-linked species to cell-surface proteins that either remained trapped in the cell membrane or led to intracellular delivery. MALDI-TOF MS is thus a powerful tool to dissect CPP internalization mechanisms.

Sequence-dependent enrichment of a model phosphopeptide: a combined MALDI-TOF and NMR study.

The goal of this study was to detect and quantify by MALDI-TOF MS the phosphorylation of a peptide containing the recognition motif of the Protein Kinase C (PKC). Such model peptide can be used as a phosphorylation probe to follow intracellular kinase/phosphatase activities. This study allowed us to establish relationships between sequence specificities and affinity for TiO(2) or IMAC media. The peptide has the sequence biotin-GGGGCFRTPSFLKK-NH(2) in which the serine residue can be phosphorylated. Enrichment of the corresponding phosphopeptide, by the dedicated IMAC and TiO(2) affinity chromatography methods, proved inefficient. By combining MALDI-TOF and NMR data, we first showed that the lack of affinity of the phosphopeptide for TiO(2) was partly related to the basic property of its peptide sequence. Furthermore, the peptide shows local structuration around the P(9)- S(10) segment, with formation of a salt bridge between the guanidinium group of the R(7) side chain and the phosphate moiety. In conjunction with an inadequate position of the {biotin-G(4)} N-terminal tag, this local structure could shield the phosphate group, preventing interaction with TiO(2). To improve TiO(2) affinity, the peptide sequence was modified accordingly. The new sequences retained the biological properties while their enrichment by IMAC or TiO(2) became possible.

Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production.

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C(10)OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

Racemic beta-sheets as templates of relevance to the origin of homochirality of peptides: lessons from crystal chemistry.

The origin of life is a historical event that has left no relevant fossils; therefore, it is unrealistic to reconstruct the chronology of its occurrence. Instead, by performing laboratory experiments under conditions that resemble the prebiotic world, one might validate feasible reaction pathways and reconstruct model systems of artificial life. Creating such life in a test tube should go a long way toward removing the shroud of mystery over how it began naturally. The riddle of the appearance of natural proteins and nucleic acids--that is, biopolymers wholly consisting of homochiral subunits (L-amino acids and D-sugars, respectively)--from the unanimated racemic prebiotic world is still unsolved. There are two hypotheses concerning the sequence of their emergence: one maintains that long homochiral (isotactic) peptides must have been formed after the appearance of the first living systems, whereas the other presumes that such biopolymers preceded the primeval enzymes. The latter scenario necessitates, however, the operation of nonlinear synthetic routes, because the polymerization of racemates in ideal solutions yields chains composed of residues of either handedness. In this Account, we suggest applying lessons learned from crystal chemistry, in which molecules from isotropic media are converted into crystals with three-dimensional (3D) periodic order, to understand how the generation of homochiral peptides from racemic alpha-amino acids might be achieved, despite its seemingly overwhelming complexity. We describe systems that include the self-assembly of activated alpha-amino acids either in two-dimensional (2D) or in 3D crystals, followed by a partial lattice-controlled polymerization at the crystal-aqueous solution interface. We also discuss the polymerization of mixtures of activated hydrophobic racemic alpha-amino acids in aqueous solutions, as initiated by primary amines or thiols. The distribution of the diastereomeric oligopeptides was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MS/MS with monomers enantioselectively tagged with deuterium. The reaction performed in aqueous solutions encompasses the following sequential steps: (i) formation of a library of short racemic peptides enriched with isotactic diastereoisomers during the early stages of the polymerization, and (ii) self-assembly of oligopeptides into racemic beta-sheet colloidal-like aggregates that are delineated by enantiotopic sites or rims; these operate as templates (nuclei) for regio-enantioselective growth in the ensuing steps of chain elongation. Desymmetrization of the racemic mixtures of peptides was achieved with enantiopure alpha-amino acid esters as initiators. The enantiomeric excess of the isotactic peptides, not including the initiator, varies with chain length, the result of a cross-enantiomeric impeding mechanism. Our results suggest a feasible scenario in which primitive homochiral peptides might have emerged early in the prebiotic world.

Dual role of hydrophobic racemic thioesters of alpha-amino acids in the generation of isotactic peptides and co-peptides in water; implications for the origin of homochirality.

Thioesters of alpha-amino acids are considered as plausible monomers for the generation of the primeval peptides. DL-Leucine-thioethyl esters (LeuSEt), where the L-enantiomer was tagged with deuterium atoms, undergo polycondensation in water or in bicarbonate or imidazole buffer solutions to yield mainly heterochiral (atactic) peptides and diketopiperazine, as analyzed by MALDI-TOF and ESI mass-spectrometry. In variance, when polymerization of DL(d(10))-Leu, first activated with N,N'-carbonyldiimidazole, then initiated with ethanethiol or with DL(d(3))-LeuSEt yielded a library of peptides up to 30 detectable residues where those of homochiral sequence (isotactic) are the dominant diastereoisomers. At these conditions, racemic beta-sheets are formed and operate as stereoselective templates in the process of chain-elongation. Isotopic L:L(d(10))-Leu co-peptides were obtained in the polymerization of L(d(10))-Leu with L-LeuSEt. By contrast, mixtures of oligo-D-Leu and oligo-L(d(10))-Leu were obtained in the polymerization of mixtures of D-LeuSEt with activated L(d(10))-Leu. Isotactic co-peptides containing Leu and Val residues were formed in the polymerization of mixtures of activated DL(d(8))-Val with DL(d(3))-LeuSEt in water, implying that the racemic beta-sheets exert regio-enantio-selection but not chemo-selection. A reaction pathway is suggested, where LeuSEt operates both as initiator of the reaction as well as a multimer.

Kinetic and structural characterization of therapeutic albumin chemical functionalization using complementary mass spectrometry techniques.

Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reaction implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conjugates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC-MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionalization kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC-MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystallography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.

Input of serum haptoglobin fucosylation profile in the diagnosis of hepatocellular carcinoma in patients with non-cirrhotic liver disease.

BACKGROUND: Haptoglobin bifucosylated tetra-antennary glycan have been identified in patients with early stage hepatocellular carcinoma, but its specificity according to the presence or not of cirrhosis has never been assessed. The aims of this study were to determine if haptoglobin bifucosylated tetra-antennary glycan (1) could be a marker of HCC in patients without cirrhosis; (2) could increase the performance of standard alpha-fetoprotein (AFP) or recent blood tests for HCC detection, i.e., lectin-reactive alpha-fetoprotein (AFP-L3), des-gamma-carboxy prothrombin (DCP) and Liver-Cancer-Risk-test (LCR1-test). METHODS: We retrospectively selected patients, 102 with HCC (21 without cirrhosis), matched by stages with 140 controls without HCC (81 without cirrhosis). Haptoglobin fucosylation was assessed by MALDI-TOF. LCR-glycan algorithm was constructed combining components of the LCR-1 test (haptoglobin, gammaglutamyl-transpeptidase, apolipoproteinA1, alpha-2-macroglobulin) with AFP, AFP-L3, DCP and haptoglobin bifucosylated tetra-antennary glycan. RESULTS: In 102 patients without cirrhosis (21 HCC and 81 controls), the intention-to-diagnose analyses showed that haptoglobin bifucosylated tetra-antennary glycan alone had a sensitivity of 71% (15/21;95%CI 50-86), significantly better (P=0.02) than standard AFP (43%;9/21;95%CI 24-63), and a specificity of 96% (78/81;95% 90-99). The sensitivity of LCR-glycan, in patients without cirrhosis, was 86% (18/21; 95%CI 63-95) significantly better (P=0.001) than standard AFP (43%; 9/21; 95%CI 24-63), with an AUROC of 0.943 (95%CI 0.806-0.98) compared to 0.811 (95%CI 0.630-0.908) for AFP (P=0.06). CONCLUSION: Haptoglobin bifucosylated tetra-antennary glycan is associated with the presence of HCC in patients with chronic liver disease including those without cirrhosis. Its combination with existing HCC biomarkers could improve the performance of standard AFP for HCC detection.

Free cholesterol transfer to high-density lipoprotein (HDL) upon triglyceride lipolysis underlies the U-shape relationship between HDL-cholesterol and cardiovascular disease.

BACKGROUND: Low concentrations of high-density lipoprotein cholesterol (HDL-C) represent a well-established cardiovascular risk factor. Paradoxically, extremely high HDL-C levels are equally associated with elevated cardiovascular risk, resulting in the U-shape relationship of HDL-C with cardiovascular disease. Mechanisms underlying this association are presently unknown. We hypothesised that the capacity of high-density lipoprotein (HDL) to acquire free cholesterol upon triglyceride-rich lipoprotein (TGRL) lipolysis by lipoprotein lipase underlies the non-linear relationship between HDL-C and cardiovascular risk. METHODS: To assess our hypothesis, we developed a novel assay to evaluate the capacity of HDL to acquire free cholesterol (as fluorescent TopFluor® cholesterol) from TGRL upon in vitro lipolysis by lipoprotein lipase. RESULTS: When the assay was applied to several populations markedly differing in plasma HDL-C levels, transfer of free cholesterol was significantly decreased in low HDL-C patients with acute myocardial infarction (-45%) and type 2 diabetes (-25%), and in subjects with extremely high HDL-C of >2.59 mmol/L (>100 mg/dL) (-20%) versus healthy normolipidaemic controls. When these data were combined and plotted against HDL-C concentrations, an inverse U-shape relationship was observed. Consistent with these findings, animal studies revealed that the capacity of HDL to acquire cholesterol upon lipolysis was reduced in low HDL-C apolipoprotein A-I knock-out mice and was negatively correlated with aortic accumulation of [3H]-cholesterol after oral gavage, attesting this functional characteristic as a negative metric of postprandial atherosclerosis. CONCLUSIONS: Free cholesterol transfer to HDL upon TGRL lipolysis may underlie the U-shape relationship between HDL-C and cardiovascular disease, linking HDL-C to triglyceride metabolism and atherosclerosis.

In-source fragmentation of very labile peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Synthetic acidic proline-rich peptides devoid of basic chemical groups were studied by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). Their ion mass spectra recorded in reflector positive ion mode have shown unusual features, i.e., absence or very weak presence of protonated peptide together with a major peak associated with fragmentation at a site that corresponds to the amide bond N-terminal to the first proline of the XPP motif. In contrast, arginine-containing analogues were stable in MALDI-TOF, whereas peptides sharing a free N-terminal amino group were moderately subject to the same fragmentation. Effects of extraction delay time suggest that this process takes place very early (nanoseconds) at the beginning of the plume expansion. The effect of the nature of the matrix on the survival yield indicates a better correlation with the initial axial velocity than with the matrix proton affinity. All the data show some strong differences with the classical in-source decay (ISD). Our results suggest the role of the available protons in the close neighborhood of the peptide during the crystallization process and the prompt fragmentation induced by collisions in the first step of ablation. Undoubtedly, our study highlights that the MALDI-TOF analysis of peptides containing proline and no basic group should be carried out with extreme caution.

D-amino acid detection in peptides by MALDI-TOF-TOF.

Detection of a D-amino acid residue in natural peptides by mass spectrometry remains a challenging task, as this post-translational modification does not induce any change in molecular mass. To our knowledge, the present article is the first report using matrix-assisted laser desorption/ionization (MALDI) for the discrimination and the quantification of peptide isomers. In this work, we used synthetic hepta- and decapeptides of biological relevance and their isomers. All-L sequences and some isomers containing a D-residue in various positions were analyzed.

Oligopeptides and copeptides of homochiral sequence, via beta-sheets, from mixtures of racemic alpha-amino acids, in a one-pot reaction in water; relevance to biochirogenesis.

As part of our studies on the biochirogenesis of peptides of homochiral sequence during early evolution, the formation of oligopeptides composed of 14-24 residues of the same handedness in the polymerization of dl-leucine (Leu), dl-phenylalanine (Phe), and dl-valine (Val) in aqueous solutions, by activation with N, N'-carbonyldiimidazole and then initiation with a primary amine, in a one-pot reaction, was demonstrated by MALDI-TOF MS using deuterium enantio-labeled alpha-amino acids. The formation of long isotactic peptides is rationalized by the following steps occurring in tandem: (i) creation of a library of short diasteroisomeric oligopeptides containing isotactic peptides in excess in comparison to a binomial kinetics, as a result of an asymmetric induction exerted by the N-terminal residue of a given handedness; (ii) precipitation of the less soluble racemic isotactic penta- and hexapeptides in the form of beta-sheets that are delineated by homochiral rims; (iii) regio-enantiospecific chain elongation occurring heterogeneously at the beta-sheets/solution interface. Polymerization of l-Leu with l-isoleucine (Ile) or l-Phe with l- (1) N-Me-histidine yielded mixtures of copeptides containing both residues. In contrast, in the polymerization of the corresponding mixtures of l- + d-alpha-amino acids, the long oligopeptides were composed mainly from oligo- l-Leu and oligo- d-Ile in the first system and oligo- d-Phe in the second. Furthermore, in the polymerization of mixtures of hydrophobic racemic alpha-amino acids dl-Leu, dl-Val, and dl-Phe and with added racemic dl-alanine and dl-tyrosine, copeptides of homochiral sequences are most dominantly represented. Possible routes for a spontaneous "mirror-symmetry breaking" process of the racemic mixtures of homochiral peptides are presented.