Mass spectrometry-based proteomics in molecular diagnostics: discovery of cancer biomarkers using tissue culture

Abstract

Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.

Figure 1

An overview of biomarker discovery using tissue culture. Cancer cells are cultured in plates. The CM as well as cells is collected separately. Extracted proteins from each fraction are processed for either in-gel or in-solution digestion followed by the detection of peptides by mass spectrometric approach. Data analysis leads to detection of candidate biomarkers.

Figure 2

(a) A schematic representation of ESI-MS—solvent along with sample flows from the needle with electrical potential generating charged droplets. The droplets carry the sample, and they are desolvated by applying heat and nebuliser gas to produce ions. These ions are now separated according to m/z ratio in the mass analyzer and registered by detector. (b) A schematic representation of MALDI-MS—the sample is mixed with the matrix and allowed to crystalize on the MALDI plate, when the laser hits the sample-matrix mixture on the plate, matrix absorbs the energy of the laser to get vaporized along with samples. Next, the charge exchange takes place from matrix and sample ions are generated.

Figure 3

An outline of 2-dimensional gel electrophoresis (2-DE). The extracted proteins are solubilized in rehydration buffer. The proteins are immobilized on IPG strips of different pH ranges depending on the requirement of the experiment. In the first-dimension, the proteins are separated on the basis of their isoelectric points (pI) and are further resolved according to their molecular weight in the second-dimension. Finally, protein spots of interest are excised and subjected to tryptic digestion followed by MS.

Figure 4

An outline figure of 2D-DIGE. Proteins are extracted from the samples and are labelled with different fluorophores as Cy 3 for sample 1, Cy 5 for sample 2, and Cy 2 for the pooled internal standard. All the samples are resolved in the same 2D gel followed by protein spot pattern detection by scanning the gel in respective wavelength for the Cy dyes; the merging of all of them yields an overlay image consisting of all three Cy dyes. The images are analyzed to get potential candidates of interest.

Figure 5

A schematic overview of SILAC. Cells are grown in normal and heavy amino acids containing media for 6 generations to achieve maximal incorporation of heavy amino acids. The proteins are extracted from both populations of cells and mixed in equal proportion and then subjected to either in-gel or in-solution digestion. Relative abundance of the digested peptides is determined from the ratio of heavy-to-light peptide signals as obtained from MS.

Figure 6

Experimental outline of iTRAQ. Proteins are extracted from either tissue samples or cultured cells and subjected to proteolytic digestion. The digested peptides are then labelled with isobaric tags followed by the pooling of the samples. The samples are then fractioned through SCX followed by tandem mass spectrometry analysis.