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 |
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