70 SPECTROSCOPY
Post-harvest chemical stable isotope labelling
Osama Chahrour and John Malone look at post-harvest chemical stable isotope labelling methods in mass spectrometry-based quantitative proteomics.
Osama Chahrour et John Malone donnent un aperçu des méthodes de marquage chimique avec des isotopes stables après récolte dans la protéomique quantitative basée sur la spectroscopie de masse.
Osama Chahrour und John Malone untersuchen chemisch stabile Isotopenmarkierungsmethoden bei der quantitative Proteomik auf der Basis der Massenspektrometrie.
S
Isotope-coded affinity tags (ICATs) are biotinylated iodoacetamide derivatives (or acrylamide derivatives) that react with the sulfhydryl groups of denatured peptide side chains. Te intermediate bridge of these reagents, contains the isotopic carbon or hydrogen atoms used for differential labelling, connecting iodoacetamide to a biotin group for affinity capture of the derivatised peptides onto a streptavidin based column.
Fig. 1. ICAT reagents structure and reactivity.
table isotopes quantitative proteomics identify equivalent peptides or peptide fragments utilising the specific increase in mass due to mass tags with stable isotopes. Te common workflow is to tag protein or peptide samples with equivalent reagents, one of which includes a heavy mass tag and the other a light mass or no tag. Te labelled samples are analysed by mass spectrometry after being mixed and fractionated. Te peaks in the mass spectra reveal the ratio of the two different isotopic or mass tag variants. Te ratio is then used to identify protein or peptide relative abundances. Several variants of the technique can be implemented, which are presented below1, 2
.
Te biotin-streptavidin affinity capture allows cysteine- containing peptides to be isolated from the complex sample mixture, thereby considerably reducing the number of different peptides/molecules introduced into the mass spectrometer ion source. Te linker bridge is available in two forms, one normal/light version and one heavy form, in which hydrogen or carbon atoms are replaced by deuterium/13C.
Two protein mixtures representing two different cell states (sick cell vs healthy cell) are treated with the light (healthy) or heavy (sick) ICAT tags (Fig. 2). Te labelled protein mixtures are then quantitatively combined and proteolysed.
Peptides binding an ICAT tag are selectively isolated by streptavidin columns and analysed by mass spectrometer. Te relative abundance of the healthy/sick states of the peptide is determined by the ratio of signal intensities of the heavy/ light tagged peptide pairs3
. Te
major disadvantage of ICAT reagents is that, approximately 10% of proteins do not have a cysteine residue and therefore are excluded from this type of analysis1,4
.
Te ICPL technology incorporates isotope labelling of free amino groups of intact proteins. ICPL reagents label lysine side chains based on the unique ability of N-hydroxysuccinimide (NHS)- nicotinic acid ester derivatives to fully derivatise primary amino groups. Terefore, the method is suitable for different kinds of protein samples, including tissues extracts or body fluids. Similar in principle to ICAT labelling, ICPL reagents also have variants with different numbers of deuterium atoms to allow multiplex quantitative analysis. For example, the ICPL Quadruplex method used ICPL_0, ICPL_4 (4 Deuteriums), ICPL_6 (6 13C) and ICPL_10 (4 Deuteriums + 6 13C) reagents allowing the simultaneous quantitation of four independent samples1
. Non-isobaric tagging, i.e. the
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