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.
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.
Oehlers
Leon P
LP
Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, 419 Centennial Hall, Texas State University San Marcos, San Marcos, TX 78666, USA.
Perez
Amy N
AN
Walter
Ronald B
RB
D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry*; D001923:Brain Chemistry / Q000502:physiology; D064026:Calbindins; D024402:Carbonic Anhydrase II / Q000378:metabolism; D030541:Databases, Genetic; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006868:Hydrolysis; D002534:Hypoxia, Brain / Q000378:metabolism*; D017879:Isothiocyanates / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D009990:Oryzias / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013385:Succinate Dehydrogenase / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
D001557:Benzenesulfonates; D064026:Calbindins; D006454:Hemoglobins; D017879:Isothiocyanates; D009419:Nerve Tissue Proteins; D010455:Peptides; D064030:S100 Calcium Binding Protein G; C015308:4-isothiocyanatobenzenesulfonate; D013385:Succinate Dehydrogenase; D005982:Glutathione Transferase; D014357:Trypsin; D005634:Fructose-Bisphosphate Aldolase; D024402:Carbonic Anhydrase II