Recent progress in protein profiling of clinical tissues for next-generation molecular diagnostics

Abstract

Differences in protein levels and the activation of signaling pathways have been extensively studied in tumor tissues, but the implementation of protein profiling methods in routine hospital workflows lags far behind that of nucleic acid-based approaches. In this review, major technologies that are currently used for measuring protein abundances in human tissues are highlighted, and for each method several examples are provided. We differentiate between extract-based and section-based methods that are each further divided into targeted- and discovery-based approaches (i.e., when the proteins to be analyzed are known or for finding promising new biomarker candidates, respectively). Current problems in protein profiling are addressed and ways in which protein profiling can successfully be implemented in routine clinical workflows are shown.

Keywords:

• antibody
• cancer
• formalin fixed and paraffin embedded
• mass spectrometry
• matrix-assisted laser desorption/ionization-imaging mass spectrometry
• personalized medicine
• reverse phase protein array
• tissue

Financial & competing interests disclosure

This work was supported by the German Federal Ministry of Education and Research (BMBF) Spitzencluster Muenchen m4 Biobank Alliance grant no. 01EX1020D. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues

• Proteins play an essential role in connecting disease genotypes with phenotypes and may help to understand patient-to-patient variabilities in treatment response or resistance.

• Tumor tissues will (also) be categorized according to deregulated pathways and networks as determined by protein profiling technologies.

• Protein profiling methods can first be divided into extract-based and section-based procedures and then into targeted and discovery-based approaches.

• Extract-based (mass spectrometry, selected reaction monitoring, Forward and reverse phase protein arrays) and section-based (tissue microarrays and matrix-assisted laser desorption/ionization-imaging mass spectrometry) protein profiling technologies offer valuable tools for proteomic research on tumor tissue.

• Mass spectrometry -based technologies offer the possibility to identify de novo biomarkers and are mainly a discovery-based tool.

• Selected reaction monitoring can be used for the quantification of 50–100 proteins per analysis with high reproducibility and without the need for specific protein binders.

• Reverse phase protein array is a highly sensitive method that can be used for the detection of low abundance proteins in limited amounts of tumor tissue, for example, biopsies.

• Due to inappropriate or delayed sample handling, protein (especially phosphoprotein) levels may change prior to fixation and stabilization in resection specimens compared with biopsies.

• The pre-analytical phase must be standardized to identify the most robust protein biomarkers for routine clinical use.

• Upcoming global standards will help to improve the pre-analytical phase for more reliable protein profiling results.

Notes