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S De Boeck

Publications and source records attributed to S De Boeck.

At least 19 recordsLinked to original sources
PubMed2001

Protein identification based on matrix assisted laser desorption/ionization-post source decay-mass spectrometry.

Due to its very short analysis time, its high sensitivity and ease of automation, matrix-assisted laser desorption/ionization (MALDI)-peptide mass fingerprinting has become the preferred method for identifying proteins of which the sequences are available in databases. However, many protein samples cannot be unambiguously identified by exclusively using their peptide mass fingerprints (e.g., protein mixtures, heavily posttranslationally modified proteins and small proteins). In these cases, additional sequence information is needed and one of the obvious choices when working with MALDI-mass spectrometry (MS) is to choose for post source decay (PSD) analysis on selected peptides. This can be performed on the same sample which is used for peptide mass fingerprinting. Although in this type of peptide analysis, fragmentation yields are very low and PSD spectra are often very difficult to interpret manually, we here report upon our five years of experience with the use of PSD spectra for protein identification in sequence (protein or expressed sequence tag (EST)) databases. The combination of peptide mass fingerprinting and PSD and analysis described here generally leads to unambiguous protein identification in the amount of material range generally encountered in most proteome studies.

Peptide Fragments↗
PubMed1999

Does dietary iron intake influence the iron status in hospitalised elderly patients?

PURPOSE: To investigate whether there is a relationship between dietary iron intake and body iron store in hospitalised elderly. METHODS: During a three months period, 163 consecutive patients older than 70 years and irrespective of their medical diagnosis participated in this study. Eight patients were excluded because of a lack of co-operation or a too bad general condition. Total food intake data were collected at admission by using a 7 days food-intake and nutrient-supplement record. The average daily dietary intake of each of the nutrients (total energy intake and potential inhibiting or enhancing factors such as calcium, dietary fibres and vitamin C) were calculated. Serum levels of ferritin, iron, transferrin, haemoglobin and mean corpuscular volume were also measured in the 155 study patients. RESULTS: A depleted iron store, defined as a serum ferritin level < 50 microg/L, was found in 23 patients (15%) of whom 17 were anaemic. The mean daily intake of iron (9.5 mg for the total group, 10 mg and 9.2 mg for men and women, respectively), vitamin C, calcium an dietary fibre was not significantly different between patients with depleted and adequate iron stores. The serum iron, transferrin, MCV, haemoglobin and ferritin levels were comparable between patients with a low (< 9 mg, n = 64) and normal (> or = 9 mg, n = 91) iron intake. We found no correlation between the dietary iron intake and the serum ferritin level (r = 0.06, p = 0.45), even not after exclusion of the patients with a serum ferritin level > 300 microg/L (r = 0.005, p = 0.95). CONCLUSION: We found no relationship between the dietary iron intake and biochemical indicators of the iron status in an elderly population. If no gastrointestinal or another organ-related cause might be found, iron deficiency can hardly be attributed to an impaired iron intake without a nutritional investigation. Other parameters may also play an important role in its multifactorial pathogenesis.

Aged↗
PubMed1997

Peptides adsorbed on reverse-phase chromatographic beads as targets for femtomole sequencing by post-source decay matrix assisted laser desorption ionization-reflectron time of flight mass spectrometry (MALDI-RETOF-MS).

We here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse-phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization-reflectron time of flight (MALDI-RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide-matrix mixtures. This permits high yield identification and sequence tagging by post-source-decay analysis on peptides derived from proteins only available in the femtomole range from one-dimensional (1-D) or two-dimensional (2-D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.

Acetonitriles↗
PubMed1997

Novel techniques for identification and characterization of proteins loaded on gels in femtomole amounts.

A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H218O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the 18O/16O-labeled fragmentation ions in the PSD spectra.

Acrylic Resins↗
PubMed1996

Structural analysis and identification of gel-purified proteins, available in the femtomole range, using a novel computer program for peptide sequence assignment, by matrix-assisted laser desorption ionization-reflectron time-of-flight-mass spectrometry.

A procedure is described for structural characterization and identification of proteins, purified by either one- or two-dimensional gel electrophoresis in the low picomole to femtomole range. The purified proteins are first detected in the primary gels by the sensitive reverse staining procedure described by Fernandez-Patron et al. (Anal. Biochem. 1995, 224, 203-211) and consecutively reeluted from combined get pieces and concentrated in the tip of a Pasteur pipette in a secondary gel matrix consisting of either sodium dodecyl sulfate-polyacrylamide or agarose. The concentrated proteins are in-matrix-digested and the resulting peptides are separated by reverse-phase high performance liquid chromatography (HPLC) combined with microsequencing or analyzed by matrix-assisted laser desorption ionization--time of flight--mass spectrometry. Protein identification is based on sequence homology or on the peptide mass pattern. The matching peptide sequences can additionally be verified by matching their measured post-source decay spectra with the calculated fragmentation patterns of the isobaric candidate peptides appearing on the search list. This is done by a computer program referred to as MassFrag, described in this paper. We demonstrate that it is possible to identify protein that are only available in the femtomole range and whose sequences are stored in nonredundant protein databases or nucleotide and expressed sequence tag databases.

Amino Acid Sequence↗
PubMed1995

An agarose-based gel-concentration system for microsequence and mass spectrometric characterization of proteins previously purified in polyacrylamide gels starting at low picomole levels.

An agarose-based concentration gel system is described for eluting and concentrating proteins previously purified either in one-dimensional or two-dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 microliters. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in-solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on-line electrospray mass spectrometry, allowing their identification by peptide mass fingerprinting.

Amino Acid Sequence↗
PubMed1986

Egg white lysozyme is the major protein of the hen's egg vitelline membrane.

Lysozyme accounts for 37% of the proteins of the hen's egg vitelline membrane. It can be extracted by salt solutions and purified by gel filtration on Sephadex G-50. There are no differences between the chemical and enzymic properties of egg white and vitelline membrane lysozymes. Vitelline membranes of ovarian eggs do not contain lysozyme. It is thus concluded that lysozyme is localized in the outer layer. Vitelline membranes from fertilized and unfertilized eggs contain the same amount of lysozyme; its percentage decreases after two days of incubation.

Amino Acids↗
PubMed1986

Mode of interaction between lysozyme and the other proteins of the hen's egg vitelline membrane.

Salt solutions and charged detergents are efficient solubilizing agents for ovovitelline membrane lysozyme. Reassociation experiments with chemically modified lysozymes indicate that positively charged amino acid residues of lysozyme (the epsilon-amino group of lysine and the guanidino group of arginine) are involved in the interaction with other proteins of the vitelline membrane. Exogenous proteins are adsorbed to lysozyme-free vitelline membranes, only if they have a high pI, comparable to that of lysozyme. It is concluded that the lysozyme-ovovitelline membrane interaction is predominantly ionic. An ovomucin-lysozyme complex is postulated as the major component of the outer layer of the membrane.

Animals↗
PubMed1978

A purine N1-C6 hydrolase activity in the chicken egg yolk: a vestigial enzyme?

All attempts to prove the presence of xanthine oxidase and uricase in yolk preparations failed. We were able instead to show that yolk preparations could hydrolyze the N1-C6 bond of certain purine bases. In the case of xanthine, 4-ureido-imidazole-5-carboxylic acid and 4-ureido-imidazole are formed. Activity only becomes apparent during purification. An analogous enzyme was described earlier in Clostridium cyclindrosporum. The liver and the blood plasma of actively laying hens do not contain the enzyme. A scheme for the degradation of egg ribonucleic acids is presented.

Allantoin↗
PubMed1975

Adenosine deaminase in chicken-egg yolk and its relation to homologous enzymes in liver and plasma of the adult hen.

Chicken egg yolk contains an adenosine deaminase that was investigated after purifying about 500 times. It has a pH optimum at 6.5, aKm of 6.6 times 10(-5) mol/l and an approximate molecular weight of 14000; higher molecular forms could not be detected. It was compared with the adenosine deaminases of chicken liver and blood plasma. From this comparison it is evident that this protein has undergone certain changes during the successive events leading to its final structure (secretion by the liver, transport through blood plasma to the oocytes and development of the egg): a common subunit with an approximate molecular weight of 15000 may be the basis of the physiological diversifications. Substrate specificity of the purified extracts extends to cytidine and guanosine also, although certain observations point to different enzymes being involved. Deoxyadenosine is also deaminated. Cu2+, Zn2+, and Pb2+ are inhibiting and free -SH seems essential for activity.

Adenosine Deaminase↗