Liquid Chromatography-nanoelectrospray Tandem Mass Research Articles

Chlamydophila psittaci-negative ocular adnexal marginal zone lymphomas express self polyreactive B-cell receptors.

The pathogenesis of Chlamydophila psittaci-negative ocular adnexal extranodal marginal zone lymphomas (OAEMZLs) is poorly understood. OAEMZLs are monoclonal tumors expressing a biased repertoire of mutated surface immunoglobulins. Antigenic activation of the B-cell receptor (BCR) may have a role in the pathogenesis of these lymphomas. We have analyzed the reactivity of recombinant OAEMZL immunoglobulins. OAEMZL antibodies reacted with self-human antigens, as demonstrated by enzyme-linked immunosorbent assays, HEp-2 immunofluorescence and human protein microarrays. All the analyzed recombinant antibodies (rAbs) exhibited polyreactivity by comprehensive protein array antibody reactivity and some rAbs also demonstrated rheumatoid factor activity. The identity of several reactive antigens was confirmed by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry. The tested rAbs frequently reacted with shared intracellular and extracellular self-antigens (for example, galectin-3). Furthermore, these self-antigens induced BCR signaling in B cells expressing cognate surface immunoglobulins derived from OAEMZLs. These findings indicate that interactions between self-antigens and cognate OAEMZL tumor-derived BCRs are functional, inducing intracellular signaling. Overall, our findings suggest that self-antigen-induced BCR stimulation may be implicated in the pathogenesis of C. psittaci-negative OAEMZLs.

Cryptosporidium p30, a Galactose/N-Acetylgalactosamine-specific Lectin, Mediates Infection in Vitro

Cryptosporidium sp. cause human and animal diarrheal disease worldwide. The molecular mechanisms underlying Cryptosporidium attachment to, and invasion of, host cells are poorly understood. Previously, we described a surface-associated Gal/GalNAc-specific lectin activity in sporozoites of Cryptosporidium parvum. Here we describe p30, a 30-kDa Gal/GalNAc-specific lectin isolated from C. parvum and Cryptosporidium hominis sporozoites by Gal-affinity chromatography. p30 is encoded by a single copy gene containing a 906-bp open reading frame, the deduced amino acid sequence of which predicts a 302-amino acid, 31.8-kDa protein with a 22-amino acid N-terminal signal sequence. The p30 gene is expressed at 24-72 h after infection of intestinal epithelial cells. Antisera to recombinant p30 expressed in Escherichia coli react with an approximately 30-kDa protein in C. parvum and C. hominis. p30 is localized to the apical region of sporozoites and is predominantly intracellular in both sporozoites and intracellular stages of the parasite. p30 associates with gp900 and gp40, Gal/GalNAc-containing mucin-like glycoproteins that are also implicated in mediating infection. Native and recombinant p30 bind to Caco-2A cells in a dose-dependent, saturable, and Gal-inhibitable manner. Recombinant p30 inhibits C. parvum attachment to and infection of Caco-2A cells, whereas antisera to the recombinant protein also inhibit infection. Taken together, these findings suggest that p30 mediates C. parvum infection in vitro and raise the possibility that this protein may serve as a target for intervention.

Variability in the immunodetection of His-tagged recombinant proteins

Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo): wild type Epo (Epo wt) and Epo containing an R103A mutation (Epo R103A). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into Chinese hamster ovary (CHO) cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni 2+– nitrilotriacetic acid resin and by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometery amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti-His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods.

Detection and Quantification of Endogenous Cyclic DNA Adducts Derived from trans-4-Hydroxy-2-nonenal in Human Brain Tissue by Isotope Dilution Capillary Liquid Chromatography Nanoelectrospray Tandem Mass Spectrometry

A sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution mass spectrometric method was developed to identify and quantify the endogenous cyclic DNA adducts derived from trans-4-hydroxy-2-nonenal with 2'-deoxyguanosine (HNE-dG) in human brain tissues. Authentic and 13C and 15N stable isotope-labeled HNE-dG were synthesized to serve as standards. The in vitro HNE-modified calf-thymus DNA as well as the DNA samples isolated from human brain tissues of normal and Alzheimer's disease subjects were enzymatically digested to nucleosides in vitro with the presence of internal standard (HNE-dG-13C10, 15N5). The enzymatic digests were cleaned up by solid phase extraction. Only 1-2 microg of DNA digests was loaded on a laboratory-constructed reversed phase capillary chromatography column, and the HNE-dG adducts were separated from intact nucleosides and quantified by a high capacity ion trap mass spectrometer in the MS/MS mode. This method was able to quantify an adduct level of approximately 40 lesions/10(9) normal DNA nucleosides. The detected level of HNE-dG adducts in hippocampus/parahippocampal gyrus and inferior parietal regions of postmortem brains from AD subjects were 556 +/- 379 and 238 +/- 72 adducts per 10(9) normal nucleosides, respectively. These results were consistent with the 32P postlabeling results, which detected 400-600 adducts per 10(9) normal nucleotides in the hippocampus.