Biomedical subjects

Petra Van Damme

Publications and source records attributed to Petra Van Damme.

6 recordsLinked to original sources

PubMed2006

Protein processing and other modifications analyzed by diagonal peptide chromatography.

Diagonal peptide chromatography consists of two consecutive, identical peptide separations with in between an enzymatic or chemical alteration of the side-chain structure of selected peptides. Such selected and altered peptides acquire different chromatographic properties thereby segregating from non-altered peptides in a series of secondary peptide separations. Originally described by Brown and Hartley in 1966, we have modified the technique such that it can be used for higher throughput gel-free proteomics. Our technique is termed COmbined FRActional DIagonal Chromatography (COFRADIC) and exploits evoked differences of the hydrophobicity of peptides in reverse-phase liquid chromatography. One important advantage of COFRADIC is its versatility: by changing the alteration reaction, different classes of peptides are sorted and finally analyzed. We previously published protocols and applications for separating methionyl, cysteinyl, amino terminal and phosphorylated peptides. In this review, we assess the potential of COFRADIC for the analysis of several posttranslational modifications emphasizing on in vivo protein processing events. Additional modifications that can be analyzed include phosphorylation and N-glycosylation. The potential of COFRADIC for isolating peptides holding such modified amino acids are discussed here.

Carbon Isotopes↗

PubMed2006

AggA is required for aggregation and increased biofilm formation of a hyper-aggregating mutant of Shewanella oneidensis MR-1.

Shewanella oneidensis COAG, a hyper-aggregating mutant of MR-1, was isolated from a rifampicin-challenged culture. Compared to the wild-type, COAG exhibited increased biofilm formation on glass carrier material. The role of surface-located proteins in the process of COAG auto-aggregation was confirmed by different proteolytic treatments of the aggregates. All of the tested proteolytic enzymes resulted in deflocculation within 3 h of incubation. In order to examine the altered expression of outer-membrane proteins in COAG, membrane-enriched cell preparations were analysed by proteomics and the protein pattern was compared to that of MR-1. From the proteomics results, it was hypothesized that the agglutination protein AggA, associated with the secretion of a putative RTX protein, was involved in the hyper-aggregating phenotype. These results were confirmed with a DNA microarray study of COAG versus MR-1. An insertional mutation in the S. oneidensis COAG aggA locus resulted in loss of the hyper-aggregating properties and the increased biofilm-forming capability. The insertional mutation resulted in strongly decreased attachment during the initial stage of biofilm formation. By complementing this mutation with the vector pCM62, expressing the aggA gene, this effect could be nullified and biofilm formation was restored to at least the level of the MR-1 wild-type.

Bacterial Adhesion↗

PubMed2005

Diagonal reverse-phase chromatography applications in peptide-centric proteomics: ahead of catalogue-omics?

Diagonal electrophoresis/chromatography was described 40 years ago and was used to isolate specific sets of peptides from simple peptide mixtures such as protease digests of purified proteins. Recently, we have adapted the core technology of diagonal chromatography so that the technique can be used in so-called gel-free, peptide-centric proteome studies. Here we review the different procedures we have developed over the past few years, sorting of methionyl, cysteinyl, amino terminal, and phosphorylated peptides. We illustrate the power of the technique, termed COFRADIC (combined fractional diagonal chromatography), in the case of a peptide-centric analysis of a sputum sol phase sample of a patient suffering from chronic obstructive pulmonary disease (COPD). We were able to identify an unexpectedly high number of intracellular proteins next to known biomarkers.

Biomarkers↗

PubMed2005

The human platelet proteome mapped by peptide-centric proteomics: a functional protein profile.

Several studies have been published in which holistic approaches were used to characterise the proteome and transcriptome of human platelets. The key intent being that a deeper understanding of the normal and aberrant physiological functions of platelets can only be achieved if most biomolecular building blocks are mapped. Here we present the application of recently developed novel technologies that overcome some of the shortcomings of gel-based proteomics. Central in our approach is the so-called combined fractional diagonal chromatography (COFRADIC)-technology in which sets of representative peptides are sorted in a diagonal RP chromatographic system through a specific modification of their side chain. In this study we combined three different COFRADIC sorting techniques to analyse the proteome of human platelets. Methionyl, cysteinyl and amino terminal peptides were isolated and analysed by MS/MS. Merging the peptide identifications obtained after database searching resulted in a core set of 641 platelet proteins, which comprises the largest set identified today. In comparison to previously published platelet proteomes, we identified 404 novel platelet proteins containing a high number of hydrophobic membrane proteins and hypothetical proteins. Furthermore we discuss the observed characteristics and potential benefits of each of the different COFRADIC technologies for proteome analysis and highlight important issues that need to be considered when searching sequence databases using data obtained in peptide-centric, non-gel proteomics studies.

Animals↗

PubMed2005

Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis.

We generated a comprehensive picture of protease substrates in anti-Fas-treated apoptotic human Jurkat T lymphocytes. We used combined fractional diagonal chromatography (COFRADIC) sorting of protein amino-terminal peptides coupled to oxygen-16 or oxygen-18 differential labeling. We identified protease substrates and located the exact cleavage sites within processed proteins. Our analysis yielded 1,834 protein identifications and located 93 cleavage sites in 71 proteins. Indirect evidence of apoptosis-specific cleavage within 21 additional proteins increased the total number of processed proteins to 92. Most cleavages were at caspase consensus sites; however, other cleavage specificities suggest activation of other proteases. We validated several new processing events by immunodetection and by an in vitro assay using recombinant caspases and synthetic peptides containing presumed cleavage sites. The spliceosome complex appeared a preferred target, as 14 of its members were processed. Differential isotopic labeling further revealed specific release of nucleosomal components from apoptotic nuclei.

Amino Acid Sequence↗

PubMed2003

Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1).

Nuclear factor kappaB (NF-kappaB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses. NF-kappaB activity has long been thought to be regulated mainly by IkappaB family members, which keep the transcription factor complex in an inactive form in the cytoplasm by masking the nuclear localization signal. Nowadays, the importance of additional mechanisms controlling the nuclear transcription potential of NF-kappaB is generally accepted. We show that the mitogen-activated protein kinase inhibitors SB203580 and PD98059 or U0126, as well as a potent mitogen- and stress- activated protein kinase-1 (MSK1) inhibitor H89, counteract tumor necrosis factor (TNF)-mediated stimulation of p65 transactivation capacity. Mutational analysis of p65 revealed Ser276 as a target for phosphorylation and transactivation in response to TNF. Moreover, we identified MSK1 as a nuclear kinase for p65, since MSK1 associates with p65 in a stimulus-dependent way and phosphorylates p65 at Ser276. This effect represents, together with phosphorylation of nucleosome components such as histone H3, an essential step leading to selective transcriptional activation of NF-kappaB-dependent gene expression.

Animals↗