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) - PowerPoint PPT Presentation
Transcript of 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 ParomovChristian MuenyiWilliam L. Stone
Proteomics techniques used to identify proteins
• 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)
S #1S #2
Vehicle 2.5 mM CEES
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.
2DGE Example comparing Vehicle- vs CEES-treated human keratinocytes
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.
Results for the Protein Database search for a protein spot #543.
← chromatogram of LC/MS data
←Three homologous proteins identified with highest probability.
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.
The list of B and Y ions as they match against the experimental data (matched B ions – red, matched Y ions – blue).
MS/MS fragment ion matches for a peptide fragment of 14-3-3 protein Sigma(YLAEVATGDDK)
The mass errors of the measurement.
MS/MS data of fragment ions that matched theoretical data of database
Conclusion:
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).
