Molecular Profiling Methods and Protocols
The next revolution in molecular medicine is the application of molecular profiling to individualized patient therapy. Molecular profiling technology has advanced dramatically, particularly in the field of cancer tissue biomarkers. It is now possible to g
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1. Introduction The emerging field of tissue proteomics stated the importance of the myriads of proteins and fragments generated by tissues which are correlated with disease outcomes (1, 2) that can be targeted for more efficacious and appropriate therapy. In fact, the discovery and characterization of valuable candidate biomarkers have the benefit to select patients who are the most inclined to respond and, subsequently, develop a patient-tailored therapy (3, 4). In these efforts to identify and measure informative biomarkers from patient body fluids and tissue samples, sensitive mass spectrometry instruments coupled to bioinformatics analysis play a central role. Mass spectrometers are powerful, versatile, and analytical instruments with the ability to sequence and characterize disease-related candidate biomarkers, both qualitatively and quantitatively (5, 6) (Table 1). Independently of the mass spectrometer employed, protein extractions from tissues of interest or body fluids
Virginia Espina and Lance A. Liotta (eds.), Molecular Profiling: Methods and Protocols, Methods in Molecular Biology, vol. 823, DOI 10.1007/978-1-60327-216-2_16, © Springer Science+Business Media, LLC 2012
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Table 1 Comparison of analytical multiplex platforms for measuring the peptidome Mass spectrometry based
Protein array based
Immuno-MS
MS profiling
Suspension beads
Antibody array
Reversephase array
Multiplex measurement
Yes
Yes
Yes
Yes
Yes
High throughput
Yes
Yes
Yes
Yes
Yes
Highly sensitive
No
No
Yes
Yes
Highly quantitative
No
No
Yes
Yes
Yes
Readout of posttranslational modification
Yes
Yes
Limited
Limited
Yes
Readout of size/mass
Yes
Yes
No
No
No
Two-site antibody capture required
No
No
Yes
Yes
No
Works with single antibody
Yes
No
No
No
Yes
Attribute
Fig. 1. Proteins are extracted from various biological samples, including tissues, serum and other biological fluids, and are separated either by 1-dimensional (1D) or 2D gel electrophoresis. Proteins are digested with proteolytic enzyme (i.e., trypsin) before mass spectrometric analysis. Mass spectrometry identifies masses of the peptides and their primary structures. The mass data obtained are then used in database searches, enabling identification of proteins.
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Mass Spectrometry-Based Biomarker Discovery
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and subsequent protein fractionation represent an important and unavoidable step in the workflow for biomarker isolation and sequencing (Fig. 1). Samples obtained from polyacrylamide gels, tissues, or directly from solution are digested with a proteolytic enzyme (i.e., trypsin digestion) into peptide fragments prior to mass spectrometer analysis (Table 2). Herein, we describe the materials and procedures necessary to isolate and sequence proteins from multiple biological samples by using nanoelectrospray ionization mass spectrometry. In detail, we describe the following procedures: acetone precipitation of proteins, Coomassie and silver staining of SDS-PAGE, in-gel digestion of proteins, in-so
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