Application of proteomics to the study of pollination drops.

PREMISE OF THE STUDY: Pollination drops are a formative component in gymnosperm pollen-ovule interactions. Proteomics offers a direct method for the discovery of proteins associated with this early stage of sexual reproduction. • METHODS: Pollination drops were sampled from eight gymnosperm species: Chamaecyparis lawsoniana (Port Orford cedar), Ephedra monosperma, Ginkgo biloba, Juniperus oxycedrus (prickly juniper), Larix ×marschlinsii, Pseudotsuga menziesii (Douglas-fir), Taxus ×media, and Welwitschia mirabilis. Drops were collected by micropipette using techniques focused on preventing sample contamination. Drop proteins were separated using both gel and gel-free methods. Tandem mass spectrometric methods were used including a triple quadrupole and an Orbitrap. • RESULTS: Proteins are present in all pollination drops. Consistency in the protein complement over time was shown in L. ×marschlinsii. Representative mass spectra from W. mirabilis chitinase peptide and E. monosperma serine carboxypeptidase peptide demonstrated high quality results. We provide a summary of gymnosperm pollination drop proteins that have been discovered to date via proteomics. • DISCUSSION: Using proteomic methods, a dozen classes of proteins have been identified to date. Proteomics presents a way forward in deepening our understanding of the biological function of pollination drops.

Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps.

Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH(2)) and EMP-EF (FDVMGIIKKIAGAL-NH(2)), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

Chemical and biological characterization of four new linear cationic á-helical peptides from the venoms of two solitary eumenine wasps

Four novel peptides were isolated from the venoms of the solitary eumeninewasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/ TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry)analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumeninewasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH2) and EMP-EF (FDVMGIIKKIAGAL-NH2), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumeninewasp. These sequences have the characteristic features of linear cationic cytolyticpeptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant a-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

Accurate quantitation of standard peptides used for quantitative proteomics.

MS-based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC-MS/MS (UPLC-MS/MS) to accurately quantify peptide standards at concentrations of 0.1-10 microM. The ability for accurate quantitation of micro-molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the amounts required.

Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma.

Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([(13)C(6)]Arg or [(13)C(6)]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.