By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip® technology. For this, cryostat sections from head and neck tumors (n ‫؍‬ 57) and adjacent mucosa (n ‫؍‬ 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p ‫؍‬ 0.000029) was isolated by twodimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexinpositive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal

The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed lasermicrodissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P ‫؍‬ 3.34 ؋ 10 ؊5 and 4.6 ؋ 10 ؊5) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.

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The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.