A strategy for combining surface plasmon resonance (SPR) biomolecular interaction analysis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. Both techniques are highly complementary but need separate optimization to improve their individual specificity and sensitivity. Sensor surfaces that are optimal for kinetic analysis are not well suited for MALDI-MS and vice versa. In addition, the transfer of analyte from SPR to MS is crucial and often accompanied by sample loss. To address both of these points, a bifunctional SPR fluid cell was constructed where optimized surfaces can be used for binding studies and MS simultaneously with regard to the special need of each technique. The setup guarantees that the SPR and the loading experiment for MS are performed at identical conditions. A removable pin carries the affinity-surface-bound analyte to the mass spectrometer so that handling is minimized, avoiding analyte elution. Functionalized transfer pins can also be used independently of SPR for microaffinity capture-MS.

Background: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca 2+ -binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell typespecific, activation-and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879-41887).

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Originally published in: Analysing Gene Expression. Edited by Stefan Lorkowski, Paul Cullen. Copyright © 2003 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30488‐2 The sections in this article are Introduction How does mass spectrometry work? Ionisation of molecules Detection of ions Appearance of spectra Modes of operation Proteomic mass spectrometry Data analysis tools for mass spectroscopic experiments Proteomics servers and databases An example: Identification of an isolated and unknown protein Is quantification possible with mass spectrometry? Analysis of protein modifications Phosphorylation Glycosylation Access to protein tertiary structure Analysis of intact proteins and non‐covalent protein complexes Affinity mass spectrometry Conclusion

Originally published in: Analysing Gene Expression. Edited by Stefan Lorkowski, Paul Cullen. Copyright © 2003 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30488‐2 The sections in this article are Introduction Conventional slab gel electrophoresis Isoelectric focusing ( IEF ) Sodium dodecyl sulphate polyacrylamide gel electrophoresis ( SDS ‐ PAGE ) and Western blotting Two‐dimensional polyacrylamide gel electrophoresis (2 D ‐ PAGE ) Two‐dimensional difference gel electrophoresis (2 D ‐ DIGE ) High‐performance liquid chromatography ( HPLC ) Ion exchange chromatography ( IEC ) Size exclusion chromatography ( SEC ) Affinity chromatography Reverse phase chromatography ( RPC ) Normal phase chromatography Capillary electrophoresis ( CE ) and capillary electrochromatography ( CEC ) Multi‐dimensional separations New approaches Isotope‐coded affinity tag method ( ICAT ™) Multi‐epitope ligand‐Kartographie ( MELK ) Conclusion