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