Adapting their metabolism to varying carbon and nitrogen sources, saprophytic fungi produce an arsenal of extracellular enzymes, the secretome, which allows for an efficient degradation of lignocelluloses and further biopolymers. Based on fundamental advances in electrophoretic, chromatographic, and mass spectrometric techniques on the one hand and the availability of annotated fungal genomes and sophisticated bioinformatic software tools on the other hand, a detailed analysis of fungal secretomes has become feasible. While a number of reports on ascomycetous secretomes of, e.g., Aspergillus, Trichoderma, and Fusarium species are already available, studies on basidiomycetes have been mainly focused on the two model organisms Phanerochaete chrysosporium and Coprinopsis cinerea so far. Though an impressive number and diversity of fungal biocatalysts has been revealed by secretome analyses, the identity and function of many extracellular proteins still remains to be elucidated. A comprehensive understanding of the qualitative and quantitative composition of fungal secretomes, together with their synergistic actions and kinetic expression profiles, will allow for the development of optimized enzyme cocktails for white biotechnology.

The secretions of the salivary parotid and submandibular-sublingual (SMSL) glands constitute the main part of whole human saliva (WS) in which proline-rich proteins (PRPs) and mucins represent dominant groups. Although proteome analysis had been performed on WS, no identification of PRPs or mucins by 2-DE and MS was achieved in WS and no comprehensive analysis of both glandular secretions is available so far. The aim of this study was to compare the protein map of WS to parotid and SMSL secretions for the display of PRPs and mucins. WS and glandular secretions were subjected to 2-DE and spots were analyzed by MALDI-MS. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic PRPs as well as the proline-rich glycoprotein (PRG) could now be mapped in WS. Acidic PRPs were found equally in parotid and SMSL secretions, whereas basic PRPs and PRG were found primarily in parotid secretion. Salivary mucin MUC7 was identified in SMSL secretion. Thus, the more abundant proteins of WS can be explained mainly by mixed contributions of parotid and SMSL secretions with only few components remaining that may be derived from local sources in the oral cavity.

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b-and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 16 . This requires only small amounts of protein, provides a relatively fast identification and can easily be automated. In the second approach, fragment ion masses of one or more proteolytic peptides from a protein digest are determined, usually by electrospray ionization tandem mass spectrometry (ESI MS/MS). Database searches can then be performed with different algorithms; either by using partly interpreted data or by submitting the list of fragment ions taken from the MS/MS spectra without any data interpretation [2,3]. However, both approaches suffer from some limitations. In peptide mass fingerprinting, results obtained are not always unambiguous and the method does not cope well with protein mixtures. On the other hand,

Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1- and 2-D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI-MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin-deficient mutants, binding of H. pylori to MUC7 and gp-340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline-rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc-alpha(2)-glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2-D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.

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Laser induced liquid beam ionization/desorption mass spectrometry (LILBID-MS) is a new desorption method recently developed in our laboratory. This method allows ions to be desorbed directly from the liquid phase into the high-vacuum region of a mass spectrometer. This method has now been applied to the detection of noncovalent protein-protein complexes. The example given in this paper is the quartenary complex of human hemoglobin. For the first time, the intact hemoglobin could be detected by laser desorption mass spectrometry. Furthermore, evidence for the specificity of the complex is given. Copyright Studying noncovalent interactions of proteins with mass spectrometry is a very difficult field of research. The difficulties arise from the fragility of these complexes in the gas phase. In solution they are held together only by weak forces, mainly hydrophobic and dipole-dipole interactions. First of all the sample preparation on the target must not destroy this delicate equilibrium. Upon desorption of the ions into the gas phase, the stabilizing forces change with polar interactions, such as salt bridges and H-bonds, being responsible for the stability of the noncovalent complex. As a result, the gas-phase structure of the complexes generally differs from that in solution. This fact alone makes it difficult to detect noncovalent complexes by mass spectrometry and to correlate mass spectra with solution behavior. Moreover, these complexes tend to dissociate in the gas phase, due to thermal excitation during the desorption process and due to their internal energy. This dissociation rate must be slow enough to detect the complexes on the time-scale of the experiment, i.e. in the microsecond regime (for a time-of-flight analyzer). As a result, great care must be taken not to destroy the complexes and not to produce artefacts by the detection process. Once these problems have been solved, a broader application of mass spectrometry in biological sciences can be anticipated.Electrospray ionization mass spectrometry (ESI-MS) has been mainly used for the detection of noncovalent interactions, 1-6, although Matrix-assisted laser desorption/ ionization (MALDI) has recently been applied to a lesser extent. [7][8][9] . MALDI suffers from two main shortcomings: first the sample preparation (crystallization of the analyte/ matrix mixture) removes the aqueous environment and, secondly, the desorption process itself seems to hinder the detection of specific noncovalent interactions in most cases. A benchmark system that challenges the potential of mass spectrometry for the detection of molecular aggregation is the hemoglobin complex. Hemoglobin is an important part of mammalian blood and transports oxygen to the cells. The intact human protein consists of four polypeptide chains, two a-chains and two b-chains, and four heme groups nested therein. These subunits are held together by noncovalent interactions, mainly salt bridges and hydrophobic interactions. The structure is sensitive to the pHvalue of the solvent. Only at a neut...

Laser-induced liquid beam ionization/desorption mass spectrometry (LILBID-MS) is a new and versatile laser desorption method that has recently been developed in our group. With this technique it is possible to desorb pre-formed ions directly from the liquid phase and to analyze them in a time-of-flight mass spectrometer. To achieve this, a microscopic liquid beam is injected into a high vacuum chamber and irradiated with pulses of an infrared laser beam. The ions set free are then orthogonally accelerated into the mass analyzer. Up to now, this method has only been able to analyze alcoholic solutions using a CO 2 desorption laser. In this paper we describe the successful application of this method to aqueous solutions by using an Nd:YAG pumped LiNbO 3 optical parametric oscillator (OPO) which is tuned to the wavelength of bulk water. First results obtained with this new laser system are described in this report.