Download Proteomics techniques used to identify proteins

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)
•
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 #1
S #2
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:
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).