Since the four major Ras isoforms are 85% identical and only 188/9 residues in length, they present a significant challenge to be targeted by SRM-based proteomics. As a result, four proteases (trypsin, LysC, GluC and elastase) were evaluated for their ability to generate proteotypic Ras peptides that distinguish the Ras isoforms. Candidate peptides were selected based on their amino acid sequence, likely charge state(s), presence of chemically modifiable residues and known Ras PTMs. From the proteases tested, trypsin generated the largest number of proteotypic peptides that were amenable to SRM analysis and defined the most aspects of cellular Ras abundance. Trypsin differentiates the four major isoforms, describes wild-type Ras at positions 12/ 13 and provides redundancy with peptides shared between isoforms. Yet, a tryptic peptide that defines total Ras levels was lacking, as the Ras identical region (residues 1-85) contains only 6 arginine and lysine residues. Consequently, an elastase-derived peptide FYTLVREI (residues 156-163) may be utilised to obtain total Ras information, but would require a separate digestion.
Tryptic peptides are commonly observed as doubly charged ions, with one proton sequestered by their C-terminal basic residue whilst the other, following excitation, can migrate to energetically less favourable sites along the peptide backbone to cause dissociation (Paizs and Suhai, 2005). As the majority of selected tryptic Ras peptides do not contain a highly basic amino acid other than at their C-terminus, they are likely to reside in a single charge state. Yet, two tryptic Ras peptides contain mid-chain histidine residues: SFEDIHHYR (K-Ras) and SFEDIHQYR (H- Ras). These were detected as doubly and triply charged precursor ions that fragment into a mix of singly and doubly charged product ions. This is not ideal for SRM, as the mid-chain histidine residues may sequester the additional proton, making the peptide less amenable to fragmentation by CID. Moreover, the signal from these peptides is spread across multiple charge states. Both of these factors will reduce the sensitivity of the SRM assay, possibly explaining why these peptides were inadequately detected from in-gel digests of 10 and 25 ng of pure Ras protein.
The presence of proline residues in peptides can be advantageous in terms of sensitivity for SRM, as peptides preferentially fragment N-terminally of these residues and generate an intense y-ion signal (Schwartz and Bursey, 1992). Two selected tryptic Ras peptides contain proline residues, the H-Ras specific peptide SYGIPYIETSAK and the shared H- and N-Ras peptide, SYGIPFIETSAK. Both generated a dominant y8 ion signal, exemplified by the H-Ras peptide, where only two other fragment ions, b2 and b3, were viable choices for transitions. y7 and y6 product ions were initially chosen, but following CE optimisation, their intensities remained ~100-fold less than that for the y8 ion. As a result, this peptide is quantified using only 3 transitions.
Peptides that contain methionine, asparagine and glutamine can be chemically modified and therefore present problems for SRM-based strategies. When modified, these residues experience a change in mass. Methionine can undergo oxidation, generating methionine sulfoxide or sulfone, causing a mass increase of 15.99 or 31.99 Da, respectively (Ghesquiere and Gevaert, 2014). Asparagine and glutamine residues can undergo deamidation and be converted into isoaspartate/ aspartate and glutamate, respectively (Geiger and Clarke, 1987; Lange et al., 2008; Piszkiewicz et al., 1970). This modification causes an increase in mass of almost 1 Da, but occurs at a much lower rate for glutamine residues. Such mass changes are important, because Q1 will eject modified peptides if not specifically selected for in SRM. Chemical modifications, like different charge states, will also act to spread a peptide’s signal and reduce the sensitivity of the assay. Tryptic peptides with methionine residues were therefore discounted: DSEDVPMVLVGNK (K-Ras) and DSDDVPMVLVGNK (H- and N-Ras). As tryptophan can also be oxidised, peptides containing this residue would also be discounted, but interestingly Ras does not contain this residue. Since Ras is a small protein, it was not possible to avoid peptides that contain asparagine or glutamine: TGEGFLCVFAINNTK (H and K), QGVEDAFYTLVR (H and N) and QGVDDAFYTLVR (K(B)-Ras). Although these peptides are prone to chemical modification, the use of the PSAQ strategy ought to compensate for acquired modifications, since the heavy standard is added at the earliest possible point in the workflow, both the isotope-labelled and endogenous proteins should be equally affected by such modifications.
Fig. 3.15. Post-translational modifications of Ras. Known modifications of H-Ras (top) and K(B)-Ras (bottom). Based on (Ahearn et al., 2012).
The only point of variation would be if there are differences between the endogenous protein isolated from cells and the isotope-labelled standards, but this is largely unavoidable. Yet, non-acidic buffers were used during the purification of isotope-labelled standards and cell lysis to limit the extent of deamidation.
Ras PTMs must also be considered when selecting peptides for quantification, as modified peptides must be specifically selected if they are to be detected using SRM. While K(B)-Ras requires farnesylation to associate with the plasma membrane, the remaining isoforms require further palmitoylation to achieve membrane binding (Ahearn et al., 2012; Prior and Hancock, 2012). These lipid modifications occur in the HVR of Ras and therefore any peptide in this region was discounted. However, Ras proteins can also be modified on residues outside of this region (Fig. 3.15). From the selected peptides (Fig. 3.8), ubiquitylation of K147 will affect SYGIPYIETSAK (H-Ras) and SYGIPFIETSAK (K- and N-Ras) (Jura et al., 2006; Sasaki et al., 2011). However, the detected levels of ubiquitylated H-Ras were between 1-2% of total H-Ras, meaning this peptide should be capable of reporting accurate H-Ras levels despite being modified. Yet, the extent of K- and N-Ras ubiquitylation at K147 is unclear, but the use of this peptide can be validated if individual values for cellular K- and N-Ras match its results.
LysC, GluC and elastase were not chosen as the primary protease to acquire information on cellular Ras abundance. LysC and GluC generated peptides with multiple mid-chain highly basic residues, meaning the fragmentation of these peptides may be limited and, as described earlier, their signals will be diluted.
overlap, their signal is shared and the possibility of detecting them in a target lysate is reduced. Consequently, these proteases were only considered to complement the tryptic digest. Elastase digestion consistently generated the pan-Ras peptide FYTLVREI, while GluC generated the pan-Ras peptide DSYRKQVVIDGE. Neither of these peptides is affected by common Ras mutations and therefore suitable to define total cellular Ras. These proteases can therefore be utilised to obtain total Ras information in a separate digest alongside trypsin.
Once the Ras proteotypic peptides were chosen, suitable transitions for each peptide were selected. To ensure that each transition was genuine, only fragment ions observed in MS/MS spectra obtained using the 4000 QTRAP were considered. y-ions were given preference over b- and a-ions, since y-ions contain more of the peptide they will have a more unique mass. Where possible, a minimum of three y- ions was selected per peptide, supplemented with up to two a- or b-ions. Transitions were optimised with respect to CE in order to increase the intensity of each fragment ion and improve assay sensitivity. Following optimisation, unlabelled and isotope- labelled proteins were mixed at various ratios and subject to in-gel digestion and SRM analysis with the optimised instrument settings. All tested peptides demonstrated a linear relationship between Ras peptide abundance and signal intensity. However, the K(B)-Ras peptide, QGVDDAFYTLVR, demonstrated an interesting phenomenon, where the inclusion of a y4 ion caused the slope of the MS response to increase. This indicates that, at higher abundances the isotope-labelled y4 ion is more intense than its unlabelled counterpart. This is puzzling, but ultimately means the y4 ion cannot be employed in the SRM assay. Along with optimised instrument parameters, a 60-minute RP-HPLC linear gradient was designed. All peptides displayed unique retention times, bar, SFADINLYR and SYGIPFIETSAK, which nearly co-elute. This unfortunately necessitates the use of two independent LC runs to monitor both of these peptides when using the 4000 QTRAP, but this shouldn’t be necessary, faster and more sensitive on newer QqQ instruments. The developed assay was readily capable of detecting all Ras isoforms at 10 ng levels and is compatible with samples derived from human, mouse, rat, chicken and guinea pig tissues. In the next chapter, the SRM Ras quantification technique was applied to quantitate Ras isoform abundance in isogenic SW48 colorectal cancer cell lines, harbouring a variety of K-Ras codon mutations, as well as H-Ras and N- RasG12V mutations.