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Análisis de proteínas expresadas mediante MS Felix Elortza

Análisis de proteínas expresadas mediante MS Felix Elortza. Overview of the work done last months. 3 proteins expressed in cell free system (rabbit reticulocytes ) DEXI, GRP114 & KCNG4 A gel was run to fractionate the sample. Bands analyzed by MS (negative result)

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Análisis de proteínas expresadas mediante MS Felix Elortza

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  1. Análisis de proteínas expresadas mediante MS Felix Elortza

  2. Overview of the work done last months • 3 proteins expressed in cell free system (rabbit reticulocytes) DEXI, GRP114 & KCNG4 • A gel was run to fractionate the sample. • Bands analyzed by MS (negative result) • Proteins “wear” GST tag, so Glutation beads were used for purification. • Negative results

  3. List of clones at Manuel Fuentes Labfor IVTT systemexpression(2012-12-05)

  4. Many transmembrane proteins (7 out of 15) • 7 Transmembrane proteins (6 prots have multiple TM) • 1 Secreted protein (WAP four-disulfide core domain protein 1) • Most of them show N-glycosylation sites • Other possible PTMs • Sample preparation should be compatible with membrane protein digestion

  5. AQP8_HUMAN • 6 Transmembranespans • 4 peptides observable, including N-terminus 1, 5 (Trp containing), • 10 (Met containing), 11 (Trp containing) • - Expressed only in pancreas and colon (UNIPROT)

  6. Transmembrane protein 170A - 3 observable peptides, 2, 7 (2 met containing), 9 (met containing)

  7. DEXI: no observable peptideswhen digested by trypsin It will never be detected in regular shot-gun analysis Instead, digested with Chemotripsin shows detectable peptides

  8. Summary

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