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Detection of hypoxia-related proteins in medaka (Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Comp Biochem Physiol C Toxicol Pharmacol. 2007 Feb; 145(1):120-33.CB

Abstract

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.

Authors+Show Affiliations

Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, 419 Centennial Hall, Texas State University San Marcos, San Marcos, TX 78666, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

16905368

Citation

Oehlers, Leon P., et al. "Detection of Hypoxia-related Proteins in Medaka (Oryzias Latipes) Brain Tissue By Difference Gel Electrophoresis and De Novo Sequencing of 4-sulfophenyl Isothiocyanate-derivatized Peptides By Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry." Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP, vol. 145, no. 1, 2007, pp. 120-33.
Oehlers LP, Perez AN, Walter RB. Detection of hypoxia-related proteins in medaka (Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comp Biochem Physiol C Toxicol Pharmacol. 2007;145(1):120-33.
Oehlers, L. P., Perez, A. N., & Walter, R. B. (2007). Detection of hypoxia-related proteins in medaka (Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP, 145(1), 120-33.
Oehlers LP, Perez AN, Walter RB. Detection of Hypoxia-related Proteins in Medaka (Oryzias Latipes) Brain Tissue By Difference Gel Electrophoresis and De Novo Sequencing of 4-sulfophenyl Isothiocyanate-derivatized Peptides By Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry. Comp Biochem Physiol C Toxicol Pharmacol. 2007;145(1):120-33. PubMed PMID: 16905368.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of hypoxia-related proteins in medaka (Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. AU - Oehlers,Leon P, AU - Perez,Amy N, AU - Walter,Ronald B, Y1 - 2006/06/21/ PY - 2006/02/01/received PY - 2006/06/08/revised PY - 2006/06/15/accepted PY - 2006/8/15/pubmed PY - 2007/4/28/medline PY - 2006/8/15/entrez SP - 120 EP - 33 JF - Comparative biochemistry and physiology. Toxicology & pharmacology : CBP JO - Comp Biochem Physiol C Toxicol Pharmacol VL - 145 IS - 1 N2 - 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. SN - 1532-0456 UR - https://www.unboundmedicine.com/medline/citation/16905368/Detection_of_hypoxia_related_proteins_in_medaka__Oryzias_latipes__brain_tissue_by_difference_gel_electrophoresis_and_de_novo_sequencing_of_4_sulfophenyl_isothiocyanate_derivatized_peptides_by_matrix_assisted_laser_desorption/ionization_time_of_flight_mass_spectrometry_ DB - PRIME DP - Unbound Medicine ER -