Table 1 Overview of the most common proteomics technologies in the research of tumour invasion and metastasis
Proteomics method | Abbreviation | Basic principle | Biological Application | Advantages | Limitations |
|---|---|---|---|---|---|
Two-dimensional gel electrophoresis | 2-DE | Proteins are first resolved by their isoelectric points, and then by molecular weights | Separation of proteins in complex biological samples | High resolution Very sensitive Direct detection of post-translational modifications | Limited automation Problematic gel-to-gel reproducibility Problematic recovery of hydrophobic and large molecular weight proteins Limited dynamic range of detection |
Two-dimensional difference gel electrophoresis | 2D-DIGE | Samples are labelled with two spectrally distinct fluorescent cyanine dyes, and run on the same 2-DE gel; the two gel images corresponding to each dye scan are then overlaid, and the intensities of paired spots are compared across the gel images | Quantification of the differences in protein expression between different samples | High sensitivity Accurate quantitation Good reproducibility | Expensive fluorophores, equipment and software |
Matrix assisted laser desorption ionisation time-of-flight mass spectrometry | MALDI-TOF MS | Tryptic digests of sample proteins are co-crystallized with matrix, and spotted onto MALDI plate; ionization occurs by pulsed laser radiation primarily absorbed by the matrix, causing desorption and ionization of the analyte; the resulting peptide ions are directed into TOF mass analyzer, where peptide masses are measured by determining the time required for the ions to traverse the length of the flight tube and reach detector | Protein identification Amino-acid sequencing Determination of the type and position of post-translational modifications | Produces less raw data than other MS techniques Data are relatively easy to interpret since most peptides carry only one charge and are present as a single peak in a spectrum | Requires previous separation of protein mixture Hampered identification of small acidic and integral membrane proteins |
Multidimensional protein identification technology | MudPIT | Mixture of tryptic peptides is resolved by the microcapillary column packed with reversed-phase resin followed by strong cation exchange resin; peptides are eluted directly from the column into the mass spectrometer to be rapidly analyzed | Large-scale protein analysis of complex biological mixtures Identification of protein complexes Determination of post-translational modifications Quantitative analysis of protein expression | Detects proteins of wide range of pI, abundance and sub-cellular distribution Employed directly on crude samples Easily automated High resolving power High sensitivity | Time-consuming Requires experienced personnel Does not detect protein activity nor interactions Limited throughput Generates the vast stream of raw data |
Surface enhanced laser desorption and ionization time-of-flight mass spectrometry (ProteinChip Technology) | SELDI-TOF MS | Protein solutions are applied to the spots of ProteinChip Arrays that contain either chemically (anionic, cationic, hydrophobic, hydrophilic, or metal ion) or biochemically (immobilized antibody, receptor, DNA, enzyme, etc.) active surface retaining proteins according to their specific physicochemical properties; after adding matrix solution to bound proteins, the latter are ionized with nitrogen laser and their molecular masses measured by TOF mass analyzer. As a result, unique protein abundance profiles of species bound to the chip surface are obtained. | Biomarker discovery Characterization of protein-protein and protein-DNA interactions and post-translational modifications (glycosylation and phosphorylation) | Suitable for crude biological samples (body fluids, cells) High-throughput capability High sensitivity Detects proteins with molecular weights lower than 6-kDa High precision and reproducibility | Additional MS analysis needed for determining the identity of differentially expressed protein species |
Isotope-coded affinity tags | ICAT | Two different protein samples are labelled at cysteines with the isotopically light and heavy ICAT reagents, combined and digested with trypsin; ICAT-labeled peptides are isolated by avidin affinity chromatography and analyzed by HPLC coupled to a tandem mass spectrometer; the ratio of ion intensities from co-eluting ICAT-labeled pairs permits the quantification, while a subsequent MS/MS scan provides the protein identification | Sequence identification and quantification of proteins in complex mixtures Analysis of protein changes in specific subcellular fractions | Selects only cysteine-containing peptides and thus effectively reduces the complexity of the peptide mixtures | Incomplete proteome coverage (10-20% of the whole cell proteome) |
Laser-capture microdisscetion | LCM | A stained tissue slide is placed under a microscope, and a specific thermoplastic polymer film is placed over the tissue; the cells of interest are shot by an infrared laser pulse, which melts and fuses the film around the targeted cells; the cells embedded in the polymer are lifted away from the remaining tissue | Isolation of pure cell populations from heterogeneous tissue sections prior to proteomic analyses focused on the investigation of novel biomarkers and drug targets | High-throughput Reduces sample heterogeneity Increases the specificity of signals obtained in downstream protein analysis | Requires competency in identifying the cells of interest Limited timeframe for microdissecting fresh frozen tissue. |
Reverse-phase protein microarrays | RPMA | Cell lysates are arrayed on nitrocellulose-coated glass slides binding denatured proteins; the slide is probed with a single antibody specific for an antigen of interest; upon signal development and imaging, the relative proportion of the analyte protein molecules can be compared between test samples on the array | Functional mapping of known cell-signalling networks or pathways Characterization of protein-protein, protein-DNA, and/or protein-RNA interactions | High-throughput Requires low sample volume Extremely sensitive analyte detection Good reproducibility, sensitivity, and robustness | The lack of availability of high-quality, specific antibodies Hampered analysis of low-abundance post-translational events |