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Table 1 Overview of the most common proteomics technologies in the research of tumour invasion and metastasis

From: Metastasis: new perspectives on an old problem

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