Marc Goethals

Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

Chromatographic Isolation of Methionine-containing Peptides for Gel-free Proteome Analysis Identification Of More Than 800 Escherichia Coli Proteins

A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis. A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching. The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures. Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins. Next to highly abundant proteins, we also detected low copy number components such as the E. coli lactose operon repressor, illustrating the high dynamic range. The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated. The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc., adding a new dimension to future proteome research.

Fusogenic Properties Of The C-Terminal Domain Of The Alzheimer Beta-Amyloid Peptide

A series of natural peptides and mutants, derived from the Alzheimer β-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29-40 and 29-42) and a central domain (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) of the β-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire β-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29-40 and 29-42 β-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29-42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 β-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the α-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the α-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29-40 and 29-42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the β-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.

An actin-binding site containing a conserved motif of charged amino acid residues is essential for the morphogenic effect of villin

The actin-binding protein villin induces microvillus growth and reorganization of the cytoskeleton in cells that do not normally produce this protein. Transfection of mutagenized villin cDNAs into CV-1 cells was used to show that a conserved, COOH-terminally located cluster of charged amino acid residues (KKEK) is crucial for the morphogenic activity of villin in vivo. In vitro experiments with a 22 amino acid synthetic peptide corresponding to this region of villin provide evidence that this motif is part of an F-actin-binding site that induces G-actin to polymerize. Chemical cross-linking of actin to this peptide, the effects of amino acid substitutions in peptides, and the behavior of villin variants further corroborate the participation of the KKEK sequence in actin contacts.

Selecting protein N-terminal peptides by combined fractional diagonal chromatography

In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines—which can include stable isotope labeling—occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography—tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ∼5 d.

Redox Proteomics of Protein-bound Methionine Oxidation

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.

Analysis of Protein Processing by N-terminal Proteomics Reveals Novel Species-specific Substrate Determinants of Granzyme B Orthologs

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.

In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.

beta-amyloid peptide interacts specifically with the carboxy-terminal domain of human apolipoprotein E: Relevance to Alzheimer's disease

Abstract : Growing evidence indicates the involvement of apolipoprotein E (apoE) in the development of late‐onset and sporadic forms of Alzheimers disease, although its exact role remains unclear. We previously demonstrated that β‐amyloid peptide (Aβ) displays membrane‐destabilizing properties and that only apoE2 and E3 isoforms inhibit these properties. In this study, we clearly demonstrate that the carboxy‐terminal lipid‐binding domain of apoE (e.g., residues 200‐299) is responsible for the Aβ‐binding activity of apoE and that this interaction involves pairs of apoE amphipathic α‐helices. We further demonstrate that Aβ is able to inhibit the association of the C‐terminal domain of apoE with lipids due to the formation of Aβ/apoE complexes resistant to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. On the contrary, the amino‐terminal receptor‐binding domain of apoE (e.g., residues 129‐169) is not able to form stable complexes with Aβ. These data extend our understanding of human apoE‐dependent binding of Aβ by involving the C‐terminal domain of apoE in the efficient formation of apoE/Aβ complex.

Plasmin Produces an E-Cadherin Fragment That Stimulates Cancer Cell Invasion

Abstract Matrix metalloproteases from the cell surface cleave an 80 kDa Ecadherin fragment (sECAD) that induces invasion of cancer cells into collagen type I and inhibits cellular aggregation. Conditioned media from MDCKts.srcCl2 cells at 40 C and 35 C, PCm.src5 and COLO-16 cells at 37 C contained spontaneously released sECAD; these 48 h old conditioned media were capable of inhibiting Ecadherin functions in a paracrine way. Here we show direct cleavage of the extracellular domain of Ecadherin by the serine protease plasmin. sECAD released by plasmin inhibits Ecadherin functions as evidenced by induction of invasion into collagen type I and inhibition of cellular aggregation. This functional inhibition by sECAD was reversed by aprotinin or by immunoadsorption on protein Sepharose 4 fast flow beads with antibodies against the extracellular part of Ecadherin. Our results demonstrate that plasmin produces extracellular Ecadherin fragments which regulate Ecadherin function in cells containing an intact Ecadherin/ catenin complex.