Forest M. White

Phosphoproteome Analysis of Capacitated Human Sperm EVIDENCE OF TYROSINE PHOSPHORYLATION OF A KINASE-ANCHORING PROTEIN 3 AND VALOSIN-CONTAINING PROTEIN/p97 DURING CAPACITATION

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.

Androgen Receptor Phosphorylation REGULATION AND IDENTIFICATION OF THE PHOSPHORYLATION SITES

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.

Electrospray Ionization Fourier Transform Ion Cyclotron Resonance at 9.4 T

We present the first results from a new electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer operated at a magnetic field of 9.4 T (i.e. > or = 2.4 T higher than for any prior FTICR instrument). The 9.4 T instrument provides substantially improved performance for large molecules (> or = 50% increase in mass resolving power) and complex mixtures (> or = 100% increase in dynamic range) compared to lower-field (< or = 6 T) instruments. The higher magnetic field makes possible larger trapped-ion population without introduction of significant space--charge effects such as spectral peak shift and/or distortion, and coalescence of closely-spaced resonances. For bovine ubiquitin (8.6 kDa) we observe accurate relative isotopic abundances at a signal-to-noise ratio greater than 1000:1, whereas a complete nozzle-skimmer dissociation electrospray ionization (ESI) FTICR mass spectrum of bovine carbonic anhydrase (29 kDa) is achieved from a single scan with a signal-to-noise ratio of more than 250:1. Finally, we are able to obtain mass resolving power, m/delta m > 200,000, routinely for porcine serum albumin (67 kDa). The present performance guides further modifications of the instrument, which should lead to significant further improvements.

Application of micro-electrospray liquid chromatography techniques to FT-ICR MS to enable high-sensitivity biological analysis

A microbore electrospray (ESI) injection system has been adapted to our 9.4-tesla ESI FT-ICR mass spectrometer, greatly enhancing the stability and sensitivity of the system. Spray was generated from micro-ESI needles made from sharply tapered, polished fused silica capillaries of 25 to 50 µm inner diameter. Micro-ESI permits low-level sample analysis by constant infusion at sub-µL/min flow rate over a wide range of solvent conditions in both positive- and negative-ion mode. The system is flexible and allows rapid conversion to allow routine LC/MS analysis on low-level mixtures presented in biological media. LC/MS analyses were accomplished by replacing micro-ESI needles with capillaries packed with reverse phase retention media to permit analyte concentration and purification prior to analysis (micro-ESI/LC). A unique nano-flow LC pumping system was developed, capable of producing a true unsplit solvent gradient at flow rates below 1 µL/min. The micro-ESI/LC FT-ICR system produces mass spectra from a mixture of three neuroactive peptides at a concentration of 500 amol/µL (5 fmol each total loaded) in biological salts with baseline separation, signal-to-noise ratio of >10:1 and mass resolving power >5000. These results represent a reduction in detection limit by a factor of ∼2 × 106 over the best previously published LC/FT-ICR MS data.

Immunodominance Among EBV-Derived Epitopes Restricted by HLA-B27 Does Not Correlate with Epitope Abundance in EBV-Transformed B-Lymphoblastoid Cell Lines

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.

Sequencing the Primordial Soup

Proteomics is the brute force attempt to systematically identify and map the total protein complement expressed by a genome or tissue. The intracellular protein differences between whole cells (diseased vs. healthy) or between other complex biological fluids are important to understanding protein function. Once the structure and interactions of all proteins are characterized — in physiological and pathological situations — therapeutic strategies can be more easily designed. Typically, these analyses are accomplished by 2D-gel electrophoresis for separation and quantitation, followed by Edman degradation and/or mass spectrometry (MS) for protein identification. Matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF) and/or nanospray ionization-MS are routinely used to identify tryptic maps of individual gel spots. Interfacing HPLC with electrospray ionization (ESI) on a tandem mass spectrometer (MS/MS) offers an attractive alternative. When coupled with automated sample handling techniques, data-dependent scanning, and database searching, it can be used with or without preliminary protein separation (1D or 2D gels). Combining these automated capabilities and adapting the sensitivity of nanospray to an on-line nanoflow-HPLC/micro-ESI assembly interfaced with ion trap MS has resulted in an even more attractive and sensitive method for protein identification and characterization. The methodology developed can be used to analyze complex biological mixtures containing hundreds of proteins, covering a wide dynamic range of concentrations. Detection limits of approximately 50 attomoles make possible the identification of several co-migrating proteins present in a single Coomassie- or silver-stained gel spot.

Identification by Mass Spectrometry of CD8+-T-Cell Mycobacterium tuberculosis Epitopes within the Rv0341 Gene Product

ABSTRACT Identification of Mycobacterium tuberculosis proteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes from M. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a single M. tuberculosis protein that is encoded by the M. tuberculosis Rv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8+ T cells from healthy blood donors in vitro and capable of recognizing and lysing M. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification of M. tuberculosis proteins that can be evaluated as potential vaccine candidates.

Matrix-shimmed ion cyclotron resonance ion trap simultaneously optimized for excitation, detection, quadrupolar axialization, and trapping

A different symmetry is required to optimize each of the three most common Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) electric potentials in a Penning (ICR) ion trap: one-dimensional dipolar ac for excitation (or detection), two-dimensional azimuthal quadrupolar ac excitation for ion axialization, and three-dimensional axial quadrupolar dc potential for ion axial confinement (trapping). Since no single trap shape simultaneously optimizes all three potentials, many trap configurations have been proposed to optimize the tradeoffs between the three requirements for a particular experiment. A more general approach is to divide each electrode into small segments and then apply the appropriate potential to each segment. Here, we extend segmentation to its logical extreme, by constructing a “matrix-shimmed” trap consisting of a cubic trap, with each side divided into a 5 × 5 grid of electrodes for a total of 150 electrodes. Theoretically, only 48 independent voltages need be applied to these 150 electrodes to generate all three desired electric potential fields simultaneously. In practice, it is more convenient to employ 63 independent voltages due to construction constraints. Resistive networks generate the potentials required for optimal quadrupolar trapping and quadrupolar excitation. To avoid resistive loss of excitation amplitude and detected signal, dipolar excitation/detection voltages are generated with a capacitive network. Theoretical Simion 6. 0 simulations confirm the achievement of near-ideal potentials of all three types simultaneously. From a proof-of-principle working model, several experimental benefits are demonstrated, and proposed future improvements are discussed.

Weak agonist self-peptides promote selection and tuning of virus-specific T cells

Recent progress has begun to define the interactions and signaling pathways that are triggered during positive selection. To identify and further examine self‐peptides that can mediate positive selection, we searched a protein‐database to find peptides that have minimal homology with the viral peptide (p33) that activates a defined P14 transgenic TCR. We identified four peptides that could bind the restriction element H‐2Db and induce proliferation of P14 transgenic splenocytes at high concentration. Two of the four peptides (DBM and RPP) were able to positively select thevirus‐specific TCR in fetal thymic organ culture but were unable to induce clonal deletion. Reverse‐phase HPLC and mass spectrometry demonstrated that these peptides were presented by H‐2Db molecules on thymic epithelial cell lines. We also examined whether the selecting ligands altered T cell responsiveness in vitro. DBM‐selected T cells lost their ability to respond to the positively selecting ligand DBM, whereas RPP‐selected T cells only retainrd their ability to respond to high concentrations of RPP. These results demonstrate that self‐peptides that mediate positiveselection can differentially tune the activation threshold of T cells and alter the functional repertoire of T cells.

Characterization and Evolutionary Relevance of the Sperm Nuclear Basic Proteins from Stickleback Fish

We have characterized the sperm nuclear basic proteins (SNBPs) of the sticklebacks in the suborder Gasterosteoidei. The complete amino acid sequence of the protamines from Aulorhynchus flavidus, Pungitius pungitius, Gasterosteus aculeatus, (anadromous) and G. wheatlandi, as well as the sequences of the protamines of several species pairs of freshwater G. aculeatus, have been determined. Analysis of the primary structure of these proteins has shown that: a) despite the relatively low amino acid complexity and small molecular mass of these basic proteins, they are very good molecular markers at the generic level. The bootstrap parsimony analysis using their sequences provides a phylogenetic relationship for the old anadromous species of Gasterosteoidei which is identical to that obtained from morphological and behavioral analysis; b) the comparison of the sequences also suggests that protamines from the suborder Gasterosteoidei have most likely evolved from a common gene in the early Acanthopterygii by an extension of the carboxy terminal portion of the molecule; c) protamines are not good markers for recent postglacial freshwater isolates of G. aculeatus. However, in the unique case of Enos Lake (British Columbia), we have been able to detect an additional minor protamine component in the benthic forms of G. aculeatus that is not present in the limnetic forms. Thus, this new protamine must have appeared during the past 12,000 years concomitantly with the speciation of benthics and limnetics in this lake. Mol. Reprod. Dev. 57:185–193, 2000.