Direct Analysis of Protein Complexes
63
A total of 75 proteins were identified using direct analysis of the peptide mixture.
Three proteins previously shown to be part of the complex were not found. One
of the proteins has a molecular weight of 3700 Dalton and pI of 12.76. When
digested with trypsin a collection of small pep tides are produced which would be
difficult to observe with the scan ranges employed in the mass spectrometry
experiment. Two other polypeptides with molecular weights of 6600 and 6700 Da,
respectively, should have been detected in the experiment. To determine if these
polypeptides are present, we used non-data dependent tandem mass spectrometry to select several ions expected to be present from each protein. The expected
limit of detection for this experiment is in the mid-attomole range on an ion trap
mass spectrometer. Tandem mass spectra were not obtained for the expected
peptides suggesting these proteins are not present within the detection limit of
the experiment. A total of 12 proteins of the ribosomal complex were identified
using liquid chromatography that were not identified in the 2-D gel experiment.
There are several potential advantages to the direct analysis approach. First protein solubility is less of a factor in the analysis of proteins. Vigorous denaturation
is possible as well as the use of detergents and organic solvents to solubilize the
proteins. Second the technique is less sensitive to the molecular weight of a protein as long as pep tides are produced that are within the scan range of the mass
spectrometer. Third the process is fully automated. Potential pitfalls of the
approach encompass the digestion of the protein and the complexity of the peptide mixture produced. Since the method requires the creation of peptides within
the scan range of the mass spectrometer, very acidic or very basic proteins can
produce pep tides too large or too small, respectively. An analysis of the yeast
genome shows 98 % of the proteins in the genome will create peptides in the
mass range of 800-2800 Dalton after proteolysis with trypsin.
9
Conclusions
Unraveling the mechanisms of biological processes will require fast and sensitive
analytical technology. Traditional protein analysis depends on gel electrophoresis
to isolate proteins prior to sequence analysis. This step potentially compromises
efficient protein recovery by requiring extraction of the protein from the polyacrylamide gel matrix. By digesting proteins in solution and analyzing the resulting complex mixture of peptides using tandem mass spectrometry, less material
is lost during sample manipulation. The ability to directly identify the components of protein mixtures allows the analysis of the proteins localized to subcellular spaces, proteins immunoprecipitated, proteins interacting in affinity interaction chromatography, and protein complexes. As technologies and methodologies improve the identification and quantification of proteins in total celllysates
will be possible.
63
A total of 75 proteins were identified using direct analysis of the peptide mixture.
Three proteins previously shown to be part of the complex were not found. One
of the proteins has a molecular weight of 3700 Dalton and pI of 12.76. When
digested with trypsin a collection of small pep tides are produced which would be
difficult to observe with the scan ranges employed in the mass spectrometry
experiment. Two other polypeptides with molecular weights of 6600 and 6700 Da,
respectively, should have been detected in the experiment. To determine if these
polypeptides are present, we used non-data dependent tandem mass spectrometry to select several ions expected to be present from each protein. The expected
limit of detection for this experiment is in the mid-attomole range on an ion trap
mass spectrometer. Tandem mass spectra were not obtained for the expected
peptides suggesting these proteins are not present within the detection limit of
the experiment. A total of 12 proteins of the ribosomal complex were identified
using liquid chromatography that were not identified in the 2-D gel experiment.
There are several potential advantages to the direct analysis approach. First protein solubility is less of a factor in the analysis of proteins. Vigorous denaturation
is possible as well as the use of detergents and organic solvents to solubilize the
proteins. Second the technique is less sensitive to the molecular weight of a protein as long as pep tides are produced that are within the scan range of the mass
spectrometer. Third the process is fully automated. Potential pitfalls of the
approach encompass the digestion of the protein and the complexity of the peptide mixture produced. Since the method requires the creation of peptides within
the scan range of the mass spectrometer, very acidic or very basic proteins can
produce pep tides too large or too small, respectively. An analysis of the yeast
genome shows 98 % of the proteins in the genome will create peptides in the
mass range of 800-2800 Dalton after proteolysis with trypsin.
9
Conclusions
Unraveling the mechanisms of biological processes will require fast and sensitive
analytical technology. Traditional protein analysis depends on gel electrophoresis
to isolate proteins prior to sequence analysis. This step potentially compromises
efficient protein recovery by requiring extraction of the protein from the polyacrylamide gel matrix. By digesting proteins in solution and analyzing the resulting complex mixture of peptides using tandem mass spectrometry, less material
is lost during sample manipulation. The ability to directly identify the components of protein mixtures allows the analysis of the proteins localized to subcellular spaces, proteins immunoprecipitated, proteins interacting in affinity interaction chromatography, and protein complexes. As technologies and methodologies improve the identification and quantification of proteins in total celllysates
will be possible.