Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleate.
The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30 kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH 6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins.
Ortsäter
Henrik
H
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. henrik.ortsater@sodersjukhuset.se
Sundsten
Tea
T
Lin
Jian-Man
JM
Bergsten
Peter
P
D000818:Animals; D002021:Buffers; D002478:Cells, Cultured; D021983:Cyclophilins / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005947:Glucose / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D019301:Oleic Acid / Q000494:pharmacology*; D010100:Oxygen / Q000032:analysis; D019696:Peptidylprolyl Isomerase / Q000302:isolation & purification / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D040081:Protein Array Analysis; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000494:pharmacology
D002021:Buffers; D007328:Insulin; D010710:Phosphates; D011506:Proteins; C081786:cyclophilin B; D019301:Oleic Acid; D014357:Trypsin; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; D005947:Glucose; D010100:Oxygen