CPSA Digest 2003

Plenary

The Multi-Dimensionality of the Proteome

The Multi-Dimensionality of the Proteome
A first-generation proteomics experiment

• defines the primary sequences of expressed proteins

A second-generation experiment seeks to gains information on:

• sub-cellular localization of proteins
• their response to external stimuli
• temporal variation in protein expression

The third-generation experiment is incompletely recognized at the present time. It is concerned with gaining information on:

• the variation in the nature, extent and kinetics of post-translational modification
• protein conformation, 3-dimensional structure and protein/protein interactions
• protein dynamics

One cannot claim to have defined the proteome unless the dynamics of individual protein interaction within that proteome can be defined.

A Typical Proteomics Experiment
A cell lysate is subjected to an n-dimensional separation (i.e., 2-D gel). A protein fraction is eluted or isolated from the separation which is then subjected to enzymatic hydrolysis. The digest is evaluated by conventional mass spectrometry (MS) where a particular protein can be recognized (via database searching) by comparison of its experimental mass profile with that predicted for a known or expected protein sequence. Sometimes not enough information is obtained for an unequivocal identification of a protein, so tandem MS is used to give additional sequence information in conjunction with database searching.

To monitor the changes in a rat hepatocyte proteome as a result of treatment in culture with a drug known to induce phospholipidosis is an example of a typical proteomics experiment. A comparison of two gels -- a control and a treated gel‹yields some information about relative patterns of proteins and provides a first impression of relative quantification. However, since a narrow range of pH 5-6 is used to improve the resolution of the gel, the scope of the evaluation is reduced to a small subset of the proteome.

Challenges and Perspectives in Analytical Sensitivity
The challenge faced in a proteomics experiment is to identify all of the protein spots in a gel. Some of these proteins are very faint and represent very low quantities of proteins. Therefore, a very sensitive analytical technique is required. Limits exist in the ability to detect, identify and recognize each protein present.

There are three perspectives to consider with regard to the sensitivity of tandem mass spectrometry (MS/MS) and MS in general:

1. Historical
2. Contextual
3. Fundamental

Historical Perspective
An example presented from 1990 used fast atom bombardment of adipokinetic hormone in Drosophila to identify a pair of ions separated by two mass units. Evaluation by MS/MS allowed much of the sequence to be inferred and greatly improved the sensitivity (by four orders of magnitude) and detectability of the work.

Contextual Perspective
Is there enough sensitivity? For example, MHC peptides function as cellular barcodes to identify context of a particular cell, and can do so with only a few protein molecules resting on a cell surface. In this case, the T-cell is 105 more sensitive than a mass spectrometer. Much improvement is needed to match biological sensitivity to identify processes at the single cell level.

Fundamental Perspective
Have the limits been reached with respect to the sensitivity that can be obtained from our analytical techniques? Improvement in ion transmission through our instruments is needed. Other goals to explore include: bimolecular chemistry in the gas phase; improvements in the intensity associated with any product ion population; and generation of adequate information to recognize the peptide, and thus, the protein from which it is derived.

Quantitative Issues in Proteomics
Relative Quantification

• Changes in expression levels; differential isotope labeling of a protein (e.g., ICAT approach) is one aspect, along with metabolic labeling at the cell culture stage and peptide labeling (following protein digestion)
• Subcellular distributions; how much protein is present
• Stoichiometry of protein complexes
• Stoichiometry of post-translational modifications (PTM); critical in phosphorylation pathways which utilize signals

Absolute Quantification
When a protein is over expressed, it is ideal to determine whether production of a particular protein has increased as well as the absolute yield for generating maximum amounts of a particular protein. An example of epitope mapping of monoclonal antibodies is shown below. The cell lysate that contains the protein of interest that incorporated the epitope is subjected to proteolytic digestion. An antibody is used to remove only the proteolytic fragments that incorporated the epitope. A washing step is followed by Ag release and the epitope sequence is identified by MALDI-ToF MS or ESI-MS/MS.

An extension of this approach is to apply the principle of internal standard by using a 13C-labeled version of the epitope as a surrogate for the intact protein.

Key Analytical Challenges in Proteomics

• Enhanced determination of peptides/proteins Developments have been made in the last 5­10 yrs that have been extraordinary in extending these analytical capabilities. In the biological arena, the limits have not been reached and improvement is expected.
• Qualitative and quantitative description of post-translational modifications
• Accurate and precise determination of relative expression levels In the context of rigorous analytical criteria, improvements in accuracy and precision must be achieved and not based on differences in densitometry.
• Protein turnover and other aspects of protein dynamics This area of protein kinetics has been understudied/neglected.
• Elucidation of protein/protein interactions An important area that has not been addressed.