Jlj Joost van Dongen

Direct sample fraction deposition using electrospray in narrow-bore size-exclusion chromatography/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer characterization

A direct sample fraction deposition method was developed for off-line size-exclusion chromatography (SEC)/matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. By using electrospray, the SEC eluent, together with a suitable matrix solution added coaxially, was directly deposited on the MALDI plate. Owing to the formation of very small droplets in electrospray, solvent evaporation is much faster. The fractionation volume in narrow-bore SEC, which can directly be collected in one MALDI spot, can easily be optimized in the range of a few microlitres. In addition, fairly homogeneous sample spots were obtained. The possible influence of composition variation of the SEC effluent on the analytical results using direct fraction deposition was investigated; no substantial effects were observed. The applicability of the method was demonstrated by characterizing a broad poly(methyl methacrylate) sample. Copyright 2000 John Wiley & Sons, Ltd.

Reversible blocking of antibodies using bivalent peptide-DNA conjugates allows protease-activatable targeting

Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Here we report a generic approach to introduce protease sensitivity into antibody-based targeting by taking advantage of the intrinsic ability of antibodies to engage in multivalent interactions. Bivalent peptide ligands with dsDNA as a rigid linker were shown to effectively bridge the relatively large distance between the two antigen binding sites within the same antibody, yielding exclusively the cyclic 1u2006:u20061 antibody–ligand complex. Size exclusion chromatography and small angle X-scattering were used to study the types of complexes formed between a model antibody and peptide–dsDNA conjugates displaying 1 or 2 peptide ligands and different linker lengths. Competitive binding assays using fluorescence anisotropy revealed that the interaction between bivalent peptide–dsDNA conjugate and antibody is 500-fold stronger than that of the monovalent peptide, allowing effective blocking of the antigen binding sites in a non-covalent manner. Cleavage of the linker between the peptide epitope and the DNA by matrix metalloprotease 2 disables this strong bivalent interaction and was shown to effectively restore the binding activity of the antibody in an in vitro binding assay. The approach presented here is broadly applicable, because it takes advantage of the Y-shaped multivalent presentation of antigen binding sites common to all antibodies and could be extended to control antibody activity by other input signals.

Characterization of poly(butylene terephthalate) by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Poly(butylene terephthalate) (PBT) samples have been analyzed with size-exclusion chromatography (SEC) using a mixed solvent of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and chloroform as the mobile phase. Several matrices and different sample deposition methods have been investigated to analyze PBT with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Optimum results have been acquired by depositing PBT on top of a 2,4,6-trihydroxyacetophenone matrix. The found MALDI-TOF-MS method can be used to analyze the end group functionalities of PBT, as demonstrated with the samples at hand. By combining SEC (off-line) with MALDI-TOF-MS, absolute molecular masses of PBT can be measured, and these have been found to be considerably lower than those determined with SEC using polystyrene standards.

End-group modification of regioregular poly(3-alkylthiophene)s

An efficient and simple procedure for end-group modification nof poly(3-alkylthiophene)s is presented which can be incorporated into the nlast step of the polymerisation reaction or employed as a npost-polymerisation modification.

Superheated high-temperature size-exclusion chromatography with chloroform as the mobile phase for π-conjugated polymers

π-Conjugated polymers for organic electronic applications are often designed to aggregate or crystallize to enhance their charge carrier mobility. Their electronic properties often improve with increasing molecular weight. Determining the molecular weight of these polymers via size-exclusion chromatography (SEC), however, is complicated due to their significant aggregation in solution. We demonstrate that superheated high-temperature SEC with chloroform as the mobile phase, operated at temperatures well above the normal boiling point of the solvent, can effectively be used to determine the molecular weight for these polymers by dissolving the aggregates.

Mapping preferred sites for fluorescent labeling by combining fluorescence and MS analysis of tryptic CNA35 protein digests

HPLC-MS analysis of tryptic protein digests in combination with fluorescence detection is presented as a convenient and quantitative method to gain insight into the relative reactivity of lysine side chains. In this scheme (tandem) mass spectrometry was used for identification of the modified residue, whereas fluorescence detection allowed determination of their relative abundance. Our method identified labeling hot-spots at two flexible parts of the collagen-binding protein CNA35, positions that were consistent with all available structural and biochemical data on the collagen-binding properties of CNA35.

Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins

BACKGROUNDnGiven the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions.nnnMETHODSnBy parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of α- and β-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/β-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared.nnnRESULTSnThe α/β-ratio is a poor marker for thalassemia patients, yet deviant α/β-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval.nnnCONCLUSIONSnAlthough subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the α/β-ratio as screening parameters.