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Defensins are highly basic cationic peptides that are important components of the innate and adaptive immune response pathways. In addition, these peptides are involved in CD8+ T cell response to HIV-1, increased pulmonary infection risk among cystic fibrosis patients, upregulated levels of HNP-5 for patients with ulcerative colitis and Crohn's disease, and monitoring HNP-3 levels as a tumor classification scheme for cutaneous T cell lymphomas, and have promise in the pharmaceutical field as a new class of antibiotics. Here we present a parallel assay for the ct (HNP1-3) and [beta] (HBD1-2) classes of defensins in saliva that are naturally observed in the concentration range of 1 ng/mL to 10/[micro]g/mL. The method utilizes solid phase extraction of saliva samples combined with liquid chromatography-tandem mass spectrometry to identify and quantitate defensin targets. The approach involves limited sample manipulation and is easily amenable to automation. The saliva samples analyzed are derived from a large cohort study focused on examining the role of polymorphisms in genes of innate and adaptive immunity in modulating the response to vaccination for two gastrointestinal tract infections: typhoid and cholera. The [alpha]-defensin levels observed range from 1 to 10 [micro]g/ mL and correlate well with known active concentrations against a wide variety of pathogens. The observed concentration range for [beta]-defensins was between the detection limit and 33 ng/mL and had a sensitivity level that was comparable to immunoassay-based detection. This method is easily adapted for use in a clinical immunology setting and can be modified for other biological matrixes. This assay will facilitate examination of the production, secretion, and regulation of defensin peptides in a direct fashion to coordinate levels of these compounds with gender, age, response to vaccination, gene copy number, and oral health.

Electron-transfer dissociation (ETD) has recently been introduced as a fragmentation method for peptide and protein analysis. Unlike collisionally induced dissociation (CID), fragmentation by ETD occurs randomly along the peptide backbone. With the use of the sequences determined from the protein termini and the parent protein mass, intact proteins can be unambiguously identified. Because of the fast kinetics of these reactions, top-down proteomics can be performed using KID in a linear ion trap mass spectrometer on a chromatographic time scale. Here we demonstrate the utility of ETD in high-throughput top-down proteomics using soluble extracts of E. coli. Development of a multidimensional fractionation platform, as well as a custom algorithm and scoring scheme specifically designed for this type of data, is described. The analysis resulted in the robust identification of 322 different protein forms representing 174 proteins, comprising one of the most comprehensive data sets assembled on intact proteins to date.

One of the major unanswered questions in quantitative proteomics is that of dynamic protein turnover in the cell. Here we present a new approach to quantitative proteomies that measures the relative dynamic turnover of proteins in cellular systems. In this approach, termed synthesis/degradation ratio mass spectromelly, stable isotope labeling is employed to calculate a relative synthesis/ degradation ratio that reflects the relative rate at which [sup.13]C is incorporated into individual proteins in the cell. This synthesis/degradation ratio calculation is based on a Poisson distribution model that is designed to support high-throughput analysis. Protein separation and analysis is accomplished by utilizing one-dimensional SDS--PAGE gel electrophoresis followed by cutting the gel into a series of bands for in-gel digestion. The resulting peptide mixtures are analyzed via solid-phase MALDI LC--MS and LC--MS/MS using a tandem time-of-flight mass spectrometer. A portion of the soluble protein fraction from an E. coli K-12 strain was analyzed with synthesis/degradation ratios varying from approximately 0.1 to 4.4 for a variety of different proteins. Unlike other quantitative techniques, synthesis/degradation ratio mass spectrometry requires only a single cell culture to obtain useful biological information about the processes occurring inside a cell. This technique is highly amenable to shotgun proteomics-based approaches and thus should allow relative turnover measurements for whole proteomes in the future.

Electrospray ionization combined with ion/ion reactions in a quadrupole ion trap can be used for the direct analysis of oligonucleotide mixtures. Elements to the success of this approach include factors related to ionization, ion/ion reactions, and mass analysis. This paper deals with issues regarding the ion polarity combination, viz., positive oligonucleotides/negative charge-transfer agent versus negative oligonucleotides/positive charge-transfer agent. Anions derived from perfluorocarbons appear to be directly applicable to mixtures of positive ions derived from electrospray of oligonucleotides, in direct analogy with positive protein ions. Conditions for forming positive oligonucleotide ions devoid of adducts were more difficult to establish than for forming relatively clean negative oligonucleotide ions. A new approach for manipulating negative ion charge states in the ion trap is described and is based on use of the electric field of the positive charge-transfer agent for storage of high-mass negative ions formed during the ion/ion reaction period. Oxygen cations are shown to be acceptable for charge-state manipulation of mixed-base oligomers but induce fragmentation in polyadenylate homopolymers. Protonated isobutylene ([C.sub.4][H.sub.9.sup.+]), on the other hand, is shown to induce significantly less fragmentation of polyadenylate homopolymers.

Bacillus spores have been identified by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. Unique patterns of biomarkers were reproducibly characterized in order to tell Bacillus species members apart. Discrimination at the strain level was possible for spores of B. cereus. Protein biomarkers were characterized by MALDI analysis using spores treated with corona plasma discharge for a short time.

In a preliminary report (Bundy, J. L.; Fenselau, C. Anal Chem. 1999, 71, 1460-1463), we demonstrated the use of lectin-derivatized surfaces to capture and concentrate complex carbohydrates as well as microorganisms from sample matrixes unamenable to direct MALDI mass spectrometry. Here, we extend the work to include samples representative of a wider variety of microorganisms of importance to human health and of enveloped viruses. In this study, lectins were immobilized directly to a membrane surface via primary amines. A complementary approach was also explored, using immobilized carbohydrates to capture bacteria via microbial lectins expressed on their surfaces. The carbohydrate-based surfaces were constructed by first immobilizing streptavidin to the membrane, followed by attachment of a commercially produced biotin/carbohydrate polymer. Acid treatment of the sample prior to mass spectrometric analysis permits the observation of protein biomarkers from the captured microbial samples in the 5-20 kDa mass range. Bacteria samples were detected from physiological buffers, urine, milk, and processed chicken samples using the biocapture probes. Viral samples were detected from culture based on glycoprotein moieties desorbed directly from the surface. The carbohydratebased system provided greater sensitivity than the lectin system, possibly due to the larger number of accessible saccharide ligands on the polymer.

Differential labeling of peptides via the use of the 18O-water proteolytic labeling method has been widely adopted for quantitative shotgun proteomics studies due to its simplicity and low reagent costs. In this report, the use of immobilized trypsin in the initial digestion step, in addition to the initial digestion step, is explored as a means to minimize postlabeling back exchange of 18O-labeled peptides into the 16O form when multidimensional peptide separation methods (here, isoelectric focusing of peptides) are incorporated into the sample workflow. Examples are shown with a mixture of standard proteins and a sample from an ongoing clinical proteomics study. [ABSTRACT FROM AUTHOR]

Developing methodology for analyzing complex protein mixtures in a rapid fashion is one of the most challenging problems facing analytical biochemists today. Recent advances in mass spectrometry for the analysis of intact proteins (i.e. the top-down approach) show great promise for rapid protein identification. The ion/ion chemistry approach for the detection and identification of target proteins in complex matrices, determination of fragmentation channels as a function of precursor ion charge state, and post-translational modification characterization are discussed with particular emphasis on tandem mass spectrometry of intact proteins. [Copyright &y& Elsevier]

The proteome of human salivary fluid has the potential to open new doors for disease biomarker discovery. A recent study to comprehensively identify and catalog the human ductal salivary proteome led to the compilation of 1166 proteins. The protein complexity of both saliva and plasma is large, suggesting that a comparison of these two proteomes will provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides. To create a more comprehensive catalog of human salivary proteins, we have first compiled an extensive list of proteins from whole saliva (WS) identified through MS experiments. The WS list is thereafter combined with the proteins identified from the ductal parotid, and submandibular and sublingual (parotid/SMSL) salivas. In parallel, a core dataset of the human plasma proteome with 3020 protein identifications was recently released. A total of 1939 nonredundant salivary proteins were compiled from a total of 19 474 unique peptide sequences identified from whole and ductal salivas; 740 out of the total 1939 salivary proteins were identified in both whole and ductal saliva. A total of 597 of the salivary proteins have been observed in plasma. Gene ontology (GO) analysis showed similarities in the distributions of the saliva and plasma proteomes with regard to cellular localization, biological processes, and molecular function, but revealed differences which may be related to the different physiological functions of saliva and plasma. The comprehensive catalog of the salivary proteome and its comparison to the plasma proteome provides insights useful for future study, such as exploration of potential biomarkers for disease diagnostics.

Shotgun proteomics, where a tryptic digest of a complex proteome sample is directly analyzed by either single dimensional or multidimensional liquid chromatography tandem mass spectrometry, has gained acceptance in the proteomics community at large and is widely used in core facilities. Here we review the development in our laboratory of an alternative first-dimension separation technique for shotgun proteomics, immobilized pH gradient isoelectric focusing (IPG-IEF). The key advantages of the technology over other multidimensional separation formats (simplicity, high resolution, and high sensitivity) are discussed. The concept of using peptide pI to filter large shotgun proteomics datasets generated by the IPG-IEF technique to minimize false positives and negatives is also introduced. Finally, an account of the comparison of the technique with the established gold standard for multidimensional separation of peptides, strong cation exchange chromatography, is presented, along with the prospects for the use of peptide pI along with accurate mass measurement for the identification of peptides.

Here we present the theoretical and experimental evaluation of peptide isoelectric point as a method to aid in the identification of peptides from complex mixtures. Predicted pI values were found to match closely the experimentally obtained data, resulting in the development of a unique filter that lowers the effective false positive rate for peptide identification. Due to the reduction of the false positive rate, the cross-correlation parameters Xcorr and deltaCn from the SEQUEST program can be lowered resulting in 25% more peptide identifications. This approach was successfully applied to analysis of the soluble fraction of the E. coli proteome, where 417 proteins were identified from 1022 peptides using just 20 microg of material.