Moving forward in colorectal cancer research, what proteomics has to tell

Table 1 Advantages and disadvantages of proteomic technologies for protein profiling

Technique	Methods		Advantages		Disadvantages
2D	Separation on a gel of the protein content of a sample in two dimensions according to mass and charge; gels are stained and spot intensities in samples are compared among different gels		High separation (thousands of proteins per gel)		Low throughput laborious (one samples per gel); poor resolution for extreme masses and extremely acidic or basic proteins; no direct protein identification; large amount of starting material compared with other techniques
2D-DIGE	Measuring three samples per gel; each of them is labelled with a different fluorescent dye, and the intensities of each gel spot for each sample are measured at a wavelength specific for the label		Direct comparison of samples on one gel: better reproductibility		Low throughput (three samples per gel)
Protein microarrays	Binding of a targeted protein in one sample to spotted probes on a ‘forward’ microarray; conversely, binding of specific probes to a targeted protein in spotted samples on a ‘reverse’ microarray; detection of bound proteins by direct labelling or by labelled secondary antibodies		High throughput in terms of number of probes per (forward) array or number of samples per (reverse) array; biomarker identity or class readily known		Synthesis of many different probes necessary; identity or class of targeted proteins must be known; limited to detection of proteins targeted by the probes
SELDI-TOF MS	Selected part of a protein mixture is bound to a specific chromatographic surface and the rest washed away		High throughput; direct application of whole sample (fast on-chip sample cleanup); small amount of starting material		Unsuitable for high molecular weight proteins; limited to detection of bound proteins; lower resolution and mass accuracy than MALDI-TOF
MALDI-TOF MS	Application of a protein mixture onto a gold plate; desorption of proteins from the plate by laser energy and measurement of the protein masses; comparison of peak intensities between multiple samples		High throughput		Need for sample fractionation of complex samples; more starting material needed for sample fractionation; unsuitable for high molecular weight proteins
LC-MS-MS	Separation of a mixture of peptides (resulting from protein digestion with trypsin) by one-, two-or three-dimensional LC and measurement of peptide masses by MS-MS		Direct identification of several hundred proteins per sample by MS-MS of peptides		Low throughput; time consuming; detection by MS–MS often not comprehensive, tus complicating comparison of different samples
ICAT	Chemical tagging of proteins on cysteine residues with a heavy or light stable isotopic; after labelling samples are mixed, proteins are digested with trypsin, and labelled peptides isolated by affinity chromatography; both samples are analysed concomitantly by LC-MS-MS		Direct identification of biomarkers by MS-MS of peptides; relative quantitation; less sample complexity than with iTRAQe; MS-MS of only differentially expressed proteins		Low throughput; tagging of only cysteine-containing peptides
iTRAQ	Chemical tagging of proteins on their amine groups with stable isotopic labels of identical mass (‘isobaric’); four different labels are available for four different samples; after labelling, samples are mixed, proteins digested with trypsin and analysed concomitantly by LC-MS-MS		Direct identification of biomarkers by MS-MS of peptides; owing to isobaric labels, selection for MS-MS of the same peptide in all four samples in the same single MS run		Low throughput (four samples per run); for generating signature ion, MS-MS of all peptides in a sample is necessary; high sample complexity and limited resolution of LC (even three dimensional), confounding by co-eluting isobaric peptides