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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

Victor Paromov

Christian Muenyi

William L. Stone


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)


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.


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.


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.


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


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).


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