RESUMEN
Interspecies hybridization between the platyfish X. maculatus Jp 163 A, and the swordtail X. helleri (Sarabia), generates F(1) hybrids with pronounced melanin pigmentation. Backcrossing of F(1) hybrids with the X. helleri parent results in 25% of progeny that will spontaneously develop melanoma. We have applied proteomic methods to this Gordon-Kosswig (G-K) melanoma model to identify candidate proteins that exhibit modulated expression in fin tissue due to interspecies hybridization and progression of hybrid tissues to spontaneous melanoma. Difference Gel Electrophoresis (DIGE) was used to minimize the variability commonly observed in quantitative analyses of comparative protein samples. Following identification of up- or down-regulated protein expression by DIGE, candidate protein spots were identified by mass spectrometric sequencing. Several protein expression differences displayed in interspecies hybrids were identified and compared to distinct differences that occur upon backcrossing and progression to melanoma. These studies are important for the identification of distinct biochemical pathways involved in the variety of Xiphophorus interspecies hybrid tumor models.
Asunto(s)
Ciprinodontiformes/metabolismo , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/metabolismo , Secuencia de Aminoácidos , Aletas de Animales/metabolismo , Aletas de Animales/patología , Animales , Quimera/genética , Quimera/metabolismo , Ciprinodontiformes/genética , Progresión de la Enfermedad , Electroforesis en Gel Bidimensional/métodos , Proteínas de Peces/análisis , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Endogamia , Melanoma Experimental/genética , Melanoma Experimental/patología , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Two-dimensional fluorescence-based difference gel electrophoresis (DIGE) was used in combination with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) to identify a set of hypoxia-related biomarker proteins in medaka (Oryzias latipes) brain tissue. Each of the proteins were identified via de novo sequencing of tryptic peptides derivatized with 4-sulfophenyl isothiocyanate (SPITC), which N-terminally sulfonates peptides and promotes facile post-source decay peptide fragmentation, resulting in greatly simplified spectra consisting mainly of y-series fragment ions. We also report that addition of the non-ionic surfactant n-octyl-beta-d-glucopyranoside significantly improves SPITC-derivatized peptide recoveries. In addition, we found that a MALDI matrix consisting of the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone, diammonium citrate, and alpha-cyano-4-hydroxycinnamic acid also improves ionization of SPITC-peptides, presumably by reducing ionization suppression effects from matrix contaminants, especially sodium cations. The DIGE experiments and analyses resulted in detection of six abundant proteins and related isozymes up-regulated (>1.49, p<0.005) in hypoxic medaka brain tissues, including two hemoglobin beta subunit forms, four carbonic anhydrase 2 forms, calbindin, aldolase, succinate dehydrogenase, and glutathione-S-transferase.
Asunto(s)
Bencenosulfonatos/química , Hipoxia Encefálica/metabolismo , Isotiocianatos/química , Proteínas del Tejido Nervioso/biosíntesis , Oryzias/metabolismo , Péptidos/química , Animales , Química Encefálica/fisiología , Calbindinas , Anhidrasa Carbónica II/metabolismo , Bases de Datos Genéticas , Electroforesis en Gel de Poliacrilamida , Fructosa-Bifosfato Aldolasa/metabolismo , Glutatión Transferasa/metabolismo , Hemoglobinas/metabolismo , Hidrólisis , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Péptidos/síntesis química , Proteína G de Unión al Calcio S100/metabolismo , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Succinato Deshidrogenasa/metabolismo , Tripsina/químicaRESUMEN
The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChip. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to alpha-cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50 mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase beta tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).