Quantitation based on precursor ions

Quantification of peptides can be based on precursor ions generated from isotope incorporation in the peptides. Isotope can be enzymatically incorporated into peptides. The most common way is to introduce two atoms of ¹⁸O into the carboxylic acid group of every proteolytic peptide in a protein pool using H218

O. The incorporation of this isotope can be achieved either with trypsin, Glu-C protease, Lys-C protease and chymotrypsin (Figure 23) (61-63). This labelled pool of peptide can be compared with the same pooled control samples labelled with ¹⁶O contained in regular water. This technique has the advantage to label all peptides except the original C-terminus of the protein (33). Stewart (et al.) has described a method allowing the conservation of the labelled peptides in natural abundance water without fear of chemical back-exchange with H216

O by adjusting the pH carefully.

Figure 23: Incorporation of two atoms of ¹⁸O by reversible binding of peptides by members of serine protease family from Fenselau (et al.) (33).

Another way to analyze peptides based on precursor ions is to incorporate stable synthetic isotope as internal standards to provide absolute quantification. The first example of this technique has been made by Barr (et al.) who incorporated deuterium labelled peptides corresponding to the proteolysis counterpart of the apolipoprotein A1 present in the sample (45). This internal standard allows peptides to be quantified because they change their mass but not their chemical behaviour. This technique called AQUA (Absolute Quantification) by

Gygi (et al.) allows multiple peptides to be quantified in a single sample and these internal standards peptides are used to precisely and quantitatively measure the absolute level of protein and post-translationally modified proteins (46). Different approaches involve chemical derivation isotope incorporation. The first one has been made in 1999 by Aebersold (et al.) who introduced the Isotope-Coded Affinity Tag (ICAT) (47). The method is based on stable isotope dilution techniques coupled with tandem mass spectrometry and allows comparing two biological samples in a single analysis. An affinity reagent is covalently bound to a particular amino acid (cysteine) in all proteins present in the sample (Figure 24) and the relative abundance of protein is determined using the ratio between two similar peptides offset by 8 Daltons.

Figure 24: Structure of the cleavable Isotope-Coded Affinity Tag (cICAT) tag and labeling of

cysteine containing peptides from Leitner (et al.) (48). Cystein containing peptides are tagged with cICAT reagent which is coupled with a biotin tag. Then these tagged peptides are extracted from solution with affinity column and analyzed after cleavage of biotin group.

The proteins are then digested into peptides and the labeled peptides are purified with an affinity tag leading to a simplification of the sample mixture. The affinity tag consists of a biotin which will be retained on a streptavidin affinity matrix leading to the extraction and purification of the labeled peptides. The drawbacks of this technique include non specific binding to the matrix and incapacity to extract non cysteine containing proteins. Aebersold simplified his technique by coupling cysteines with solid beads giving a simpler, more efficient and more sensitive aspect to this approach (49). However the limitation to cysteine containing protein may compromise low level analysis. The quantification is based on the difference in mass of the tag between the control and treated experiment at the first stage MS.

Several other similar techniques have been proposed later, based on the chemical modification of proteins or peptides. Münchbach (et al.) has elaborated a similar technique to ICAT but based on tagging the peptides on their N-terminal amino acid either with H4 or with D4 reagent. The quantification is then based on the 4 Da difference in the MS spectra instead of the 8 Da or 9 Da difference for ICAT or cleavable isotope coded affinity tag (cICAT) reagents, respectively (Figure 25) (50).

Figure 25: Schematic representation of the ICAT method from Steen (et al.) (20). Samples

are tagged either with light (¹H) or heavy (²H). Then proteins are extracted and quantification is performed at the MS level where a difference of 8 Da exists between light and heavy.

Cagney modified this method by proposing his Mass-Coded Abundance Tagging (MCAT) based on differential guanidination of C-terminal lysine residue of peptides (51).

The principle is based on the modification of a lysine residue using O-methylisourea which transforms the lysine into homoarginine which is 42 Dalton heavier than lysine. Moreover, this modification does not affect the biophysical properties using LC-MS. The quantification is then performed by monitoring the ratio between modified and non-modified proteins. The

next improvement based on differential protein analysis has been made by Goodlett (et al.) who proposed a per-methyl esterification of peptides (52). The quantification was based on the difference of the D0- or D3-methanol once separated from the corresponding peptide in the second dimension of the tandem mass spectrometer (MS/MS). Another method based on the relative intensities of extracted ion chromatograms is called Isotope-Coded Protein Label (ICPL). This method is capable of high throughput quantitative proteome profiling and is based on stable isotope tagging at the frequent free amino groups of isolated intact proteins (53). Two different experimental groups are individually alkylated and differentially labeled at the free amino groups with isotope encoded (heavy) or isotope free (light) ICPL tags (53).

Figure 26 describes the workflow of this amine-based tagging.

Figure 26: Overview of the ICPL workflow from Schmidt (et al.) (53).

These stable isotope labeling techniques for proteomics are often based on complex and expensive reagents. A different approach emerged and was based on metabolic

incorporation of labeling protein into protein (54). This approach is particularly adapted to single cells grown in culture and different isotopically enriched amino acids can be used such as arginine, lysine, tyrosine and leucine. An enhanced metabolic labelling technique emerged then from specific incorporation of amino acids in cell into all mammalian proteins. This technique has been described by Ong (et al.) and is called SILAC (Stable Isotope Labelling by Amino acids in Cell culture) (55). It allows comparing two different populations of cells which will be labelled either with a normal amino acid or an isotope labelled amino acid. Cell culture media lack an essential amino acid which is replaced, with the same labelled amino acid. This labelled amino acid is then incorporated into all proteins during protein synthesis (Figure 27).

Figure 27: Schematic representation of the SILAC method from Steen (et al.) (20).

Isotopically enriched amino acid is incorporated during cell culture growth, which can be seen as a shift in mass in the MS analysis.

No chemical labelling or affinity purification steps are necessary using this technique which is compatible with all cell culture conditions, including primary cells. Ong (et al.) show that incorporation of the labelled amino acid is complete in the proteome and that cells

remain normal in the presence of the labelled media. These incorporations can be used with arginine, lysine, tyrosine and leucine (74-77).