Identification of regulatory proteins from human cells using 2d ge and lc ms ms
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Identification of regulatory proteins from human cells using 2D-GE and LC-MS/MS. Victor Paromov Christian Muenyi William L. Stone. Proteomics techniques used to identify proteins. Obtain cell lysates Run two-dimensional gel electrophoresis (2D-GE) for each sample (in triplicates)

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Identification of regulatory proteins from human cells using 2d ge and lc ms ms

Identification of regulatory proteins from human cells using 2D-GE and LC-MS/MS

Victor Paromov

Christian Muenyi

William L. Stone


Identification of regulatory proteins from human cells using 2d ge and lc ms ms

Proteomics techniques used to identify proteins 2D-GE and LC-MS/MS

  • Obtain cell lysates

  • Run two-dimensional gel electrophoresis (2D-GE) for each sample (in triplicates)

  • Compare the gels and identify over-expressed and/or under-expressed proteins compared to protein spots from control treatment

  • Identify those proteins (for each spot) by:

    • Cutting out the gel spot and trypsinizing the protein

    • Run LS/MS/MS on LTQ XL

    • Identify proteins by matching the tryptic peptides to theoretical fragments (Protein Database search)


Identification of regulatory proteins from human cells using 2d ge and lc ms ms

2DGE Example comparing Vehicle- vs CEES-treated human keratinocytes

Vehicle

2.5 mM CEES

S #2

S #1

Proteomics Study of CEES toxicity in human keratinocytes: EpiDerm tissues were exposed to vehicle or 2.5 mM CEES for 18 h. Cell lysates were separated by 2D gel electrophoresis, Coomassie blue-stained and photographed (three gels per sample). Average differences in protein expression were quantified with Dymension-2 Software. The proteins differentially expressed after CEES exposure are marked with red circles. Protein spots were excised from the gel, destained, trypsinized, and subjected to LC/MS/MS analysis.


Identification of regulatory proteins from human cells using 2d ge and lc ms ms

Results for the Protein Database search for a protein spot #543.

← chromatogram of LC/MS data

←Three homologous proteins identified with highest probability.

The protein identified belong to the 14-3-3 family of regulatory proteins.

The 14-3-3 proteins (zeta/delta, theta, and sigma) have same MW = 28 kDa and pI = 4.5.


Identification of regulatory proteins from human cells using 2d ge and lc ms ms

Included in the report is the amino acid residue coverage for 14-3-3 protein Sigma.

Top panel: protein sequence with identified peptides (shown in red).

Bottom panel: The list of identified tryptic peptides.


Identification of regulatory proteins from human cells using 2d ge and lc ms ms

MS/MS fragment ion matches for a peptide fragment of 14-3-3 protein Sigma

(YLAEVATGDDK)

The list of B and Y ions as they match against the experimental data (matched B ions – red, matched Y ions – blue).

The mass errors of the measurement.

MS/MS data of fragment ions that matched theoretical data of database


Identification of regulatory proteins from human cells using 2d ge and lc ms ms

Conclusion protein Sigma:

LC/MS/MS analysis and UniProt database search with “rigorous” filtering of the search results allow identification of three 14-3-3 regulatory proteins (zeta, theta, and sigma).