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
peer-reviewed · Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology · 2007
| Date | 2007-02-01 |
| Type | peer-reviewed |
| Venue | Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology |
| Publisher | Elsevier BV |
| Contribution | downstream-application |
| DOI | 10.1016/j.cbpc.2006.06.005 |
| Citations (OpenAlex) | 42 |
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.
Methods and tools
- Medaka brain hypoxia proteome: Identified hypoxia-responsive proteins in medaka brain by difference gel electrophoresis and de novo sequencing of SPITC-derivatized peptides by MALDI-TOF/TOF.
Cites (2)
- De novo sequencing of tryptic peptides sulfonated by 4‐sulfophenyl isothiocyanate for unambiguous protein identification using post‐source decay matrix‐assisted laser desorption/ionization mass spectrometry (2004) crossref
- A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry (1999) crossref