Protein Phosphorylation

Proteoprofile™ Trypsin

Product No: PP 0100

A solution of 2.5 mg/ml E. Coli in Cellular and Organelle Membrane Solubilizing Reagent of ProteoPrep™Total Extraction Sample Kit (Product Code Prot-Tot) was sonicated, then reduced with tributyl phosphine and alkylated with iodoacetamide (Product Code PROT-RA). 109 5g of protein was loaded on a 7 cm, pH 4-7 IPG strip (Product Code I2906), focused for 50,000 volt hours, then run on a 4-20% Tris-Glycine SDS- page gel at 150 volts for 70 min. The gel was then stained with EZBlue (G 1041). A random spot was then excised and tryptically digested using the ProteoProfile Typsin In-Gel Digest Kit (Product Code PP 0100)

The sample was desalted using a ZipTip™C18 pipette tip from Millipore and eluted directly onto the MALDI target using the MALDI matrix (a-cyano-4-hydroxycinnamic acid, 10 mg/ml in 70% ACN, 0.03% TFA). MALDI analysis was performed in the reflection positive ion mode. The resulting monoisotopic masses were searched against the NCBI database at a tolerance of 150 ppm. The protein was identified as outer membrane protein 3a from E. Coli with the matched peptides providing 30% sequence coverage.

Tryptic Fragment Amino Acid M+H+
T9 104-117 1654.8328
T10 118-124 818.4347
T10* 118-124 oxidized 834.4296
T13 135-159 2601.2949
T15 191-213 2232.1665
T19 252-263 1280.6487
T20 264-276 1409.6661
Missing Clevages Amino Acid M+H+
T20-21 264-277 1565.7572
T21-22 277-288 1378.7694