Although the human genome
has now been completely sequenced, we are still a long way from
a full understanding of the proteins encoded by these genes and
the complex biological systems in which they're involved. A new
fundamental concept called the "proteome" has recently
emerged, and proteomics is a new discipline which complements physical
genomic research, by comparing the proteomes of various structures
and systems under different conditions to further unravel biological
processes.
Basic Steps in a Proteomic Assay:
1. Purification of protein complex or subnuclear structure
2. Protein separation (1D or 2D polyacrylamide gel electrophoresis)
3. Protein identification (peptide mass fingerprinting and mass
spectrometry)**
4. Bioinformatics (cross reference of protein informatics with genomic
databases)**
5. Protein characterization (expression in bacteria and mammalian
cells, antibody generation, etc.)
** These studies are all done in collaboration with Matthias Mann
and co-workers at the Protein
Interaction Laboratory in Odense, Denmark.
To increase the amount of information
that we can extract from a proteomic screen, we have adopted
the SILAC (Stable Isotope Labeling of Amino acids in Cell
culture) approach, in which cells are metabolically labeled
through growth in medium containing a specific amino acid
with either carbon or nitrogen or both substituted with the
heavy isotopes 13C and 15N. Incorporation of these amino acid
tags allows us to determine in which cells the proteins originated.
The three cell lines can be, for example, the same cell line
under control conditions vs. two different experimental conditions,
or three different cell lines expressing three different proteins.
The diagram to the right shows the basic design of a triple
encoded SILAC experiment. After cells are grown for five passages
in the labeled media, the cells are mixed and the structure
or complex of interest either purified or immunoprecipitated.
Mass spectrometric analysis can either be performed on this
complex mix of proteins, or the proteins can be separated
on a gel, the gel cut into slices and these individual slices
subjected to mass spectrometric analysis.
The peptides that are identified are then used both to identify
the original protein and to compare and quantify levels of
labeled arginine so that we know in which cell lines(s) they
originated.
To download SILAC information and protocols, visit our Protocols
and IP SILAC Protocols pages.
For more information about SILAC, visit the CEBI
site at the University of Odense, Denmark.
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This triple-encoding SILAC approach was originally used to examine
the dynamic turnover of nucleolar proteins in mammalian cells.
In this case, the triple encoding was used to define the proteome
of purified nucleoli at various time points after transcriptional
inhibition.
"Nucleolar proteome dynamics" (2005) Andersen
JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M. Nature,
433:77-83.
| View
abstract in printable form |
Download a PDF
file of the full manuscript from Nature |
All of the data, including the kinetic data, identified in this
study is available in an online searchable database, which can
be found here:
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NoPDB: Nucleolar Protein Database
recently published as: "NoPdb: Nucleolar Proteome
Database" (2006) Leung, A.K., Trinkle-Mulcahy, L.,
Lam, Y.W., Andersen, J.S., Mann, M. and Lamond, A.I. Nucleic
Acids Res. 34:D218-20, 2006. |
More recently, this approach has been adapted for comparative
proteomics, to identify novel protein phosphatase 1 (PP1)
targeting subunits that show preferential binding for either
the α- or γ-isoform of the protein. In this case,
the affinity tag on its own was used as an internal negative
control, allowing us to rapidly separate real hits from the
large number of contaminants. For more information about PP1,
visit the
LTM site.
"Repo-Man recruits PP1γ
to chromatin and is essential for cell viability" (2006)
Trinkle-Mulcahy, L., Andersen, J., Lam, Y.W., Moorhead, G.,
Mann, M. and Lamond, A.I. J. Cell Biol. In Press. |
Other Proteomic Studies:
Over the past few years we have also used standard (i.e. non-SILAC)
proteomic approaches to identify several novel proteins in the
mammalian spliceosome complex and in the CDC5 splicing sub-complex:
"Large-scale
proteomic analysis of the human spliceosome." Rappsilber,
J., Ryder, U., Lamond A.I. and Mann, M. (2002) Genome Research,
8:1231-1245.
"Mass
Spectrometry and EST-database searching allows characterization
of the multi-protein spliceosome complex"; Neubauer, G.,
King, A., Rappsilber, J., Calvio, C., Watson, M., Ajuh, P.,
Sleeman, J., Lamond, A. and Mann, M. (1998) Nature Genetics
20, pp 46-50.
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Download a table
listing spliceosome
proteins in PDF format |
"Functional analysis of the human CDC5L complex and identification
of its components by mass spectrometry." Ajuh, P.M., Kuster,
B., Panov, K., Zomerdijk, J.C.B.M., Mann, M. and Lamond, A.I.
(2000) EMBO J. 19:6569-81.
We have also used this same type of approach to isolate both
nucleoli and Cajal bodies from mammalian nuclei and analyze
their protein compositions:
"Directed
Proteomic Analysis of the Human Nucleolus"; Andersen, J.S.,
Lyon, C.E., Fox, A., Leung, A., Lam, Y.W., Steen, H., Mann,
M. and Lamond, A.I. (2002) Current Biology, 12:1-11.
and the accompanying...
"Paraspeckles:
A novel nuclear domain"; Fox, A.H., Lam, Y.W., Leung, A.,
Lyon, C., Andersen, J., Mann, M. and Lamond, A.I. (2002) Current
Biology, 12:13-25.
Download a PDF
file of the full manuscript from Current Biology |
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"Large-scale isolation of Cajal bodies from HeLa cells."
Lam, Y.W., Lyon, C. and Lamond, A.I. (2002) Molecular Biology
of the Cell, 13:2461-2473.
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