Clinical proteomics

Proteomics technologies enable global and unbiased (i.e. not restricted to preconceived targets) view of biological systems. Great advances in mass spectrometry, computational analysis and sample preparation techniques have dramatically increased confidence in peptide identification, accuracy of protein quantification and comprehensiveness of proteomic analysis. With these advances it is now possible to apply proteomics to almost all research fields and biological systems.

The field of clinical proteomics can be divided into the analysis of tissues and body fluids. Application of proteomics to clinical samples has been a long-standing goal, aiming to find novel biomarkers and regulators of disease. So far these had been hampered by the limit on sample amount, the complexity of the biological samples and the inability to metabolically label tissue samples for quantitative analysis. In our laboratory we address multiple aspects of clinical proteomics research: Analysis of formalin fixed tissues, tissue quantification with SILAC, analysis of small amounts of tissues material and analysis of body fluids.


Proteomic analysis of formalin-fixed paraffin embedded tissue


Tissue bio-banks are an invaluable source of tumor tissues for research. For many years tissues have been fixed with formalin and embedded in paraffin (FFPE) prior to storage, in order to preserve tissue architecture. However these treatments impede global analysis of DNA, RNA and previously also proteins. We and others have developed methods to reverse the effects of formalin fixation, thereby enabling MS-based analysis of proteins and their modifications (e.g. phosphorylation and glycosylation). The FFPE-FASP method includes reversal of the formalin fixation by prolonged boiling of the tissue in an SDS-DTT buffer, similar to the antigen retrieval procedure commonly used in immunohistochemistry. This step is followed by protein digestion according to the filter aided sample preparation (FASP) technique. Digestion on top of filters ensures that highly non-reversed cross-linked peptides will not contaminate the sample. The FFPE-FASP method extends the reach of proteomics to the analysis of nearly unlimited number of fixed tissues from bio-banks.



Super-SILAC for quantification of human tumor tissues


Quantification of tumor proteomes is of prime importance in cancer research, which attempts to discover novel biomarkers and therapeutic targets. Accurate quantification of human tumor samples has been hampered by the inability to metabolically label the tissues to obtain accurate quantification. Using SILAC as a ‘spike-in’ standard rather than one of the experimental systems enables the use of heavy labeled cell lines for quantification of non-labeled tissues. The heterogeneity of tumor tissues is addressed by combining several cancer cells as a standard in the form of a super-SILAC mix. We have demonstrated highly accurate and precise breast cancer proteome quantification using the breast cancer super-SILAC mix, and we are currently developing super-SILAC mixes for multiple tumor types. The simplicity of use compared to other labeling techniques will further enable the non-proteomic specialists to use super-SILAC for their research and, we hope, for clinical applications.



 Analysis of microdissected tissue samples


Tumors are heterogenous tissues, consisting of cancer cells, stromal cells, immune cells and extracellular matrix proteins. Therefore, analysis of whole tissues averages the protein content from each of these sources, which may hinder discovery of the actual proteomics alterations in the cancer cells. Laser-capture microdissection isolates various regions of tissue slices under the microscope, and increases the homogeneity of the analyzed sample. However, typically, microdissection isolates only a few thousands of cells and up to a few micrograms of protein. We have developed a modified FASP protocol to enable efficient digestion with minimal sample loss. Addition of carrier substances such as polyethylene glycol or dextran to the processed samples improves the peptide yields, and enabled identification of thousands of proteins from sub-microgram initial protein amounts.



Analysis of body fluids- urinary proteome


Body fluids (e.g. blood, urine, cerebrospinal fluid) contain proteins that are secreted by multiple tissues and organs in the body. Changes in their proteomes thus reflect the state of the body, and can highlight disease states. Analysis of protein changes in body fluids is minimally invasivene, making it attractive for clinical diagnostics. However, analysis of serum or plasma is highly challenging due to the large dynamic range of protein abundance that spans over eleven orders of magnitude. To date, this dynamic range limits the ability to comprehensively analyze the serum proteome.

A currently more realistic task would be to analyze the urinary proteome. We have developed a method for efficient extraction of urine proteins, single-run LC-MS/MS analysis and label-free quantification of proteins. Using this method we identified over 600 proteins with high proportion of secreted, membrane and high-molecular weight proteins. This work determined the normal fluctuation of individual urinary proteins over time and between individuals, and such an approach may be useful in establishing significance thresholds for biomarker studies.


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