Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation.

A tandem quadrupole time-of-flight (Q-TOF) mass spectrometer has been programmed such that phosphorylated peptides can automatically be discovered and identified in a way similar to that of the use of precursor ion or neutral loss scanning, but without the need to scan the quadrupole mass filter. Instead, the method capitalizes on the innate capability of the Q-TOF to record mass spectra and product ion spectra quickly, with good sensitivity and with good mass accuracy. Alternate mass spectra, with and without fragmentation, are recorded at high and low collision energy with the quadrupole operating in wideband mode. The method of analysis is both compatible with and dependant on liquid chromatography for separation of complex mixtures. The method has been demonstrated by searching for the neutral loss of 98 Da (H3PO4) from phosphoserine and phosphothreonine residues, or for the phosphorylated immonium ion at m/z 216 from phosphotyrosine. The method also incorporates acquisition of the product ion spectrum from any candidate precursor ions, thereby allowing confirmation of the neutral loss or product ion and providing additional sequence information to assist identification of the protein and assign the site of phosphorylation.

High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer.

Consideration of the special problems encountered in ultra-high sensitivity biopolymer sequencing studies has led to the development of a novel quadrupole/erthogonal-acceleration time-of-flight tandem mass spectrometer described for the first time here. The performance characteristics of this new geometry are demonstrated, including fully resolved daughter-ion spectra with mass accuracies of 0.1 dalton, which allow removal of interpretation ambiguities and easy differentiation of charge states even in weak collisionally-activated decomposition tandem mass spectra. The instrument has been applied to a variety of biopolymer research problems, including the structure determination of major histocompatibility complex peptide antigens using liquid chromatography/electrospray mass spectrometry and nanoflow-electrospray tandem mass spectrometry, and sequencing capability in the low-femtomole and attomole ranges is demonstrated.

Gene structure and larval expression of cnox-2Am from the coral Acropora millepora.

We have cloned a Hox-like gene, cnox-2Am, from a staghorn coral, Acropora millepora, an anthozoan cnidarian, and characterised its embryonic and larval expression. cnox-2Am and its orthologs in other cnidarians and Trichoplax most closely resemble the Gsx and, to a lesser extent, Hox 3/4 proteins. Developmental northern blots and in situ hybridisation are consistent in showing that cnox-2Am message appears in the planula larva shortly after the oral/aboral axis is formed following gastrulation. Expression is localised in scattered ectodermal cells with a restricted distribution along the oral/aboral body axis. They are most abundant along the sides of the cylindrical larva, rare in the oral region and absent from the aboral region. These cells, which on morphological grounds we believe to be neurons, are of two types; one tri-or multipolar near the basement membrane and a second extending projections in both directions from a mid-ectodermal nucleus. Anti-RFamide staining reveals neurons with a similar morphology to the cnox-2Am-expressing cells. However, RFamide-expressing neurons are more abundant, especially at the aboral end of the planula, where there is no cnox-2Am expression. The pattern of expression of cnox-2Am resembles that of Gsx orthologs in Drosophila and vertebrates in being expressed in a spatially restricted portion of the nervous system.

Pax gene diversity in the basal cnidarian Acropora millepora (Cnidaria, Anthozoa): implications for the evolution of the Pax gene family.

Pax genes encode a family of transcription factors, many of which play key roles in animal embryonic development but whose evolutionary relationships and ancestral functions are unclear. To address these issues, we are characterizing the Pax gene complement of the coral Acropora millepora, an anthozoan cnidarian. As the simplest animals at the tissue level of organization, cnidarians occupy a key position in animal evolution, and the Anthozoa are the basal class within this diverse phylum. We have identified four Pax genes in Acropora: two (Pax-Aam and Pax-Bam) are orthologs of genes identified in other cnidarians; the others (Pax-Cam and Pax-Dam) are unique to Acropora. Pax-Aam may be orthologous with Drosophila Pox neuro, and Pax-Bam clearly belongs to the Pax-2/5/8 class. The Pax-Bam Paired domain binds specifically and preferentially to Pax-2/5/8 binding sites. The recently identified Acropora gene Pax-Dam belongs to the Pax-3/7 class. Clearly, substantial diversification of the Pax family occurred before the Cnidaria/higher Metazoa split. The fourth Acropora Pax gene, Pax-Cam, may correspond to the ancestral vertebrate Pax gene and most closely resembles Pax-6. The expression pattern of Pax-Cam, in putative neurons, is consistent with an ancestral role of the Pax family in neural differentiation and patterning. We have determined the genomic structure of each Acropora Pax gene and show that some splice sites are shared both between the coral genes and between these and Pax genes in triploblastic metazoans. Together, these data support the monophyly of the Pax family and indicate ancient origins of several introns.

Pax-6 origins--implications from the structure of two coral pax genes.

Vertebrate Pax-6 and its Drosophila homolog eyeless play central roles in eye specification, although it is not clear if this represents the ancestral role of this gene class. As the most "primitive" animals with true nervous systems, the Cnidaria may be informative in terms of the evolution of the Pax gene family. For this reason we surveyed the Pax gene complement of a representative of the basal cnidarian class (the Anthozoa), the coral Acropora millepora. cDNAs encoding two coral Pax proteins were isolated. Pax-Aam encoded a protein containing only a paired domain, whereas Pax-Cam also contained a homeodomain clearly related to those in the Pax-6 family. The paired domains in both proteins most resembled the vertebrate Pax-2/5/8 class, but shared several distinctive substitutions. As in most Pax-6 homologs and orthologs, an intron was present in the Pax-Cam locus at a position corresponding to residues 46/47 in the homeodomain. We propose a model for evolution of the Pax family, in which the ancestor of all of the vertebrate Pax genes most resembled Pax-6, and arose via fusion of a Pax-Aam-like gene (encoding only a paired domain) with an anteriorly-expressed homeobox gene resembling the paired-like class.

Novel HY peptide antigens presented by HLA-B27.

We have identified two peptides corresponding to the male-specific HY minor histocompatibility Ags presented by HLA-B27 in transgenic rodents, isolated from whole cell extracts and from immunoprecipitated B27 molecules of male B27 rat spleen cells. HPLC peptide fractions that sensitized female B27 targets for lysis by B27-restricted anti-HY CTL were analyzed by electrospray tandem mass spectrometry using a new highly sensitive quadrupole/time-of-flight instrument. Two peptide sequences were obtained, KQYQKSTER and AVLNKSNREVR. Synthetic peptides corresponding to these sequences bound B27 in vitro and were recognized by distinct B27-restricted anti-HY CTL populations. Neither peptide sequence entirely matches known protein sequences or shows a resemblance to known Y chromosome genes, but both show homology to known autosomally encoded proteins. Both peptides were shown to be controlled by the Sxr(b) segment of the short arm of the mouse Y chromosome, a segment known to contain all previously identified HY Ags. Taken together, these findings suggest that the two peptides arise as a result of Y chromosome-regulated control of one or more autosomal gene products. Although arginine at position 2 is a dominant anchor residue for peptides bound to B27, neither B27-presented HY sequence contains this residue. These studies, employing sensitive new methodology for identification of MHC-bound peptides, significantly extend the complexity of the genetic basis of HY Ags and expand the repertoire of antigenically active peptides bound to B27.