Stable Isotope Labelling Proteomics

1.6.2.1 Stable isotope labelling with amino acids in cell culture (SILAC)

In contrast to other isotope labelling techniques, SILAC is an in vivo labelling strategy in which the proteome is labelled as cells grow in culture. SILAC relies on the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium, 13C, 15N) so that two cell populations can be grown in culture media that are identical except that one of them contains a 'light' and the other a 'heavy' form of a particular amino acid (e.g. 12C and 13C labelled L-lysine, respectively) which can be readily distinguished by mass spectrometry. If the labelled analogue of an amino acid is supplied instead of the natural abundant amino acid, it will be incorporated into each newly synthesized protein chain (Ong, Blagoev et al. 2002). In fact, SILAC has the ability to compare up to five states in a single experiment, the number of states restricted by the limited availability of heavy forms of amino acids, though it can only be used in metabolically active cells so cannot be used for tissue samples (Harsha, Molina et al. 2008). It has been used to study posttranslational modifications such as protein phosphorylation and methylation, to characterise signalling pathways and to determine specific protein interactions (Gruhler and Kratchmarova 2008). The labelling process itself is uncomplicated and

highly efficient (100% of sample is available for labelling) and due to the combination of unlabelled and labelled samples prior to cell lysis it allows any method of protein or peptide purification to be used without introducing error (Ong, Foster et al. 2003).

SILAC has been used for phosphoproteomic analysis (Pimienta, Chaerkady et al. 2009), to discover differentially expressed plasma membrane proteins in renal cell carcinoma (Aggelis, Craven et al. 2009), to identify secreted proteins for pancreatic serum biomarker discovery (Yu, Barry et al. 2009), and even to help quantitatively identify proteins linked to human embryonic stem cells (Collier, Sarkar et al.), cells which could be used in the treatment of numerous medical conditions, showing its usefulness as a complementary technique for generating a list of possible protein disease markers.

1.6.2.2 Isotope-Coded Affinity Tags

The isotope coded affinity tag is a chemical modification strategy that allows quick and accurate quantification coupled with sequence identification of proteins in a complex mixture. Isotope-coded affinity tags (ICAT) and an improved version known as isobaric tags for relative and absolute quantification (iTRAQ) are the two main methods used. ICAT utilises reagents consisting of three functional groups; a sulfhydryl-reactive iodoacetate group, a biotin affinity group, and a linker carrying light or heavy isotopes (Sethuraman, McComb et al. 2004). ICAT-labelled peptides elute as pairs from a reverse-phase column so by calculating the ratio of the areas under the curve for identical peptide peaks labelled with the light and heavy ICAT reagent, the relative abundance of that peptide in each sample can be determined, which is directly related to the abundance of the corresponding protein. In addition,

because the ICAT reagents are specific for cysteinyl residues, the complexity of the original peptide mixture is greatly reduced (Li, Steen et al. 2003). Also, because it is based on post-isolation stable isotope labelling of proteins it is not limited to cells and tissues compatible with metabolic labelling.

The ICAT approach allows quantitative cataloguing and comparison of protein expression in a number of states; normal, developmental, and disease, with obvious uses in cancer biomarker discovery. The combination of ICAT and multidimensional chromatography followed by mass spectrometry of digested proteins is able to detect and quantify low abundant proteins in complex mixtures (Gygi, Rist et al. 2002).

There are a number of limitations to ICAT including missed identification of proteins with few or no cysteine residues, lost information for post-translational modifications (PTM’s), differential reversed-phase elution of identical peptides labelled with the hydrogen/deuterium isotope pairs, and complicated interpretation of tandem mass spectrometry (MS/MS) spectra due to the addition of the biotin group (Goshe and Smith 2003, Leitner and Lindner 2004), however most of these problems have been solved by the use of cleavable isotope-coded affinity tags (cICAT).

cICAT involves labelling samples with the isotopically light or heavy cICAT reagent, combining them, digesting with a protease, retrieval using an avidin affinity column and importantly having the added bonus of being able to remove the retrieval ligand prior to MS, and subsequently can be used to reduce the complexity of digested proteins and to improve quantification of low-abundant proteins (Qu, Jusko et al. 2006).

Another method using isotope affinity tags, known as iTRAQ, allows up to eight samples to be analysed in a single experiment enabling simultaneous identification and quantification, both relative and absolute, using synthetic isobaric peptide standards that are indistinguishable by their MS spectra or MS/MS ion series, but exhibit intense, low-mass MS/MS signature ions that permit quantitation of members of the multiplex set (Ross, Huang et al. 2004). The distribution of isotopes in the different tags is such that when the tags fragment a reporter ion is released which is tag-specific and the ratio of signal intensities from these tags act as an indication of the relative proportions of that peptide between the different labelled samples (Unwin). Performed in 4plex or 8plex experiments, the amine specificity of the iTRAQ reagents makes most peptides in a sample amenable to this labelling strategy.

iTRAQ, generally in conjunction with either LC-MS/MS or MALDI-TOF/TOF MS or a combination of both, has been used to find new and better biomarkers for gastric cancer (Chong, Lee et al.), to uncover clinically relevant candidate markers for prostate cancer progression (Glen, Evans et al. 2010), to study p53-modulated proteins secreted in lung cancer cells (Chenau, Michelland et al. 2009), to identify serum biomarkers for oral squamous cell carcinoma (Bijian, Mlynarek et al. 2009), to investigate low-grade breast primary tumour tissues with and without metastases and metastasis for relevant biomarkers (Bouchal, Roumeliotis et al. 2009), and to identify serum biomarkers in brain-injured patients that may predict elevated intracranial pressure (Hergenroeder, Redell et al. 2008), to name a few areas of biomarker discovery iTRAQ has been used, and is presently used, for.

There are however limitations with labelling-based quantification approaches including increased time and complexity of sample preparation, requirement for higher sample concentration, the high cost of reagents, incomplete labelling, and the requirement for specialised computer software for quantification. Also, labelling strategies limit the number of samples that can be analysed in a single experiment and some labelling strategies can not be applied to all types of samples .