Pull down
experiments/ MALDI-TOF
When a protein
has been identified as being
involved in a phenomenon, the next step is often to identify what it
interacts with. This is usually done by either (i) raising antibodies
to the known protein and then pulling the protein down from a cell
lysate, by using Sepharose- Protein A beads, which bind the Fc portion
of the antibody and easily from a pellet on centrifugation or (ii)
making
an expression construct if the cDNA for the known gene is available
such that a sequence "tag" is translated as a fusion with the gene
product. In the latter case the expression plasmid is transfected into
cells and the fusion protein and its binding partners can be spun down,
attached to beads which have a high affinity for the sequence tag.
The proteins
attached to the beads are then subjected to polyacrylamide
gel electrophoresis under denaturing conditions, and interacting
proteins may be seen after staining these gels.
Identifying such
bands used to be a difficult procedure. But the
advent
of MALDI-TOF mass spectrometry and the large sequence databases that
have accrued from various genome sequencing projects have simplified
this considerably. MALDI-TOF instruments identify peptide masses very
accurately and have high sensitivity, so very little protein is needed.
The instrument separate peptides based on the ratio of their mass to
charge (m/z) ( you don't need to know more than this). The basis
by which proteins can be uniquely identified is that the pattern of
tryptic digestion of peptides is highly specific for each protein.
Trypsin cuts C-terminal to K or R amino-acids and these are
interspersed
at different intervals in different proteins. Thus the profile of
peptide sizes measured in the MALDI-TOF instrument is highly likely to
be
unique for many proteins. These fingerprints can be computed for known
protein sequences and matched to experimental meaurements to identify
unknown proteins.
In practice an
unknown band observed in a pulldown experiment may be
cut out of the gel and eluted. Often, although a tiny amount of protein
is
involved, MALDI-TOF
mass spectrometry of the trypsinised band may identify the unique m/z
values for this protein,
which allows its subsequent identification.