Synthesis of a radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol: identification of the phosphorylation sites by N-terminal peptide sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
A novel radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol (DTT-S-C[3H(2)]CO(2)H, [3H]CM-DTT), was designed and synthesized at the micromole level by reaction of dithiothreitol with tritiated iodoacetic acid (I-C[3H(2)].CO(2)H). The reaction progress was followed by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The usefulness of the synthesized reagent was evaluated in a series of experimental approaches. (i) The synthetic phosphopeptide, NSVS(P)EEGRGDSV, was derivatized by [3H]CM-DTT separated from excess reagent by RP-HPLC. The extent of derivatization was quantitated in terms of the mol of P-Ser/mol of peptide by 3H counting, and the location of the phosphoserine was defined by the N-terminal Edman degradation sequence analysis as being the fourth amino acid residue from the N terminus. (ii) A sample of trypsin-digested alpha-casein was derivatized with [3H]CM-DTT, peptides were separated by RP-HPLC, and aliquots of each fraction were counted for 3H label within the peptide map which rapidly pinpointed the original four phosphoserine-containing peptides. This demonstrated the utility of the synthesized radioactive thiol agent in rapid purification and identification of phosphopeptides from HPLC peptide mapping of proteolytic digests of phosphoproteins. (iii) The [3H]CM-DTT was also used to determine the extent of phosphorylation of phosphoproteins both qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography and quantitatively by 3H counting. The synthesized radioactive thiol reagent [3H]CM-DTT proved to be very efficient and sensitive and should be adaptable to a wide range of routinely utilized laboratory approaches in many fields of the biological sciences.
Salih
Erdjan
E
Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopaedic Surgery, Harvard Medical School and Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011851:Radioactivity; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013439:Sulfhydryl Reagents / Q000138:chemical synthesis* / Q000737:chemistry; D014316:Tritium
C481759:1-S-(3H)carboxymethyl-dithiothreitol; D010748:Phosphopeptides; D013439:Sulfhydryl Reagents; D014316:Tritium; D010768:Phosphoserine; D004229:Dithiothreitol