Quantitative Proteomics: Increased Selectivity Decreases Interference

In quantitative proteomics, the reality of triple quadrupole instruments means that interference can occur when nearly isobaric ions are coeluted with precursor ions in the first quadropole and when the m/z values of the subsequent fragment ions are similar to those of the product ions on the third quadrupole. The level of interference this creates in analytical results varies relative to the abundance of the analyses versus the complexity of the background.

In order to evaluate the relative selectivity of SRM and LC-MS/MS analyses, Gallien et al.¹ analyzed biological samples in SRM mode on a triple quadrupole instrument and in PRM mode using a quadrupole-Orbitrap (Thermo Scientific). This side-by-side extended study modeled the gain in selectivity that researchers in the field of quantitative proteomics achieve with the increased mass resolution available with Q-TOF and quadrupole-Orbitrap instruments.

Gallien et al. analyzed 35 highly purified isotopically labeled peptides in two parallel studies: one on a triple quadrupole instrument in SRM mode and one on a quadrupole-Orbitrap mass spectrometer in PRM mode. The researchers found that the instrument in SRM mode was less selective and more likely to coelute low-abundance interferences and that, overall, lower LOQs were achieved in PRM mode. For example, when comparing the data derived from the SRM mode and the PRM mode analyses, at one dilution point in particular, twice as many transitions were quantifiable in the PRM mode. Overall, 61% of the transitions demonstrated a lower LOQ value in PRM mode, 20% evidenced identical LOQ values, and 19% had lower LOQ values in SRM mode.

The researchers also investigated two specific transitions that yielded significant differences between SRM and PRM methods. While the SRM transitions demonstrated two distinct elution profiles with coeluting interference, the mass spectrometer in PRM mode evidenced two transitions with perfectly coeluting profiles. To confirm this result, the researchers used an instrument with lower resolving power to reanalyze the PRM data. The resultant profiles were similar to those demonstrated in SRM mode, indicating the presence of interference. The full spectra measurements from the quadrupole-Orbitrap enabled researchers to detect the interfering fragment ions and extract ion chromatogram traces. By comparing the elution profiles, it was confirmed that this component, particularly in low concentrations, contributed to the SRM signal.

In terms of quantitative proteomics, a final perceived benefit of the quadrupole-Orbitrap instrument is its ability to measure fragment ions from several precursors in a single scan using the high-resolution Orbitrap. To evaluate this function further, Gallien et al. performed a series of PRM analyses of 122 isotopically labeled peptides at varying resolution powers (17,500 to 70,000) with varying selection window values between 2 and 24 m/z units. In this way, the researchers determined that as the resolving power was decreased and the width of the isolation window increased, the overall selectivity of the Orbitrap, as evidenced by increased transitions showing interference, decreased. In general, selectivity was excellent, with a 9% interference rate when the instrument was set to the highest resolving power and narrowest isolation window. At the same resolving power, an increase in the isolation window to 24 m/z resulted in over 40% interference. Overall, the loss of selectivity produced by doubling the width of the isolation window is compensated for by maintaining a doubled resolving power up to isolation windows of 16 m/z.

References

1. Gallien, S., et al., (2012) ‘Selectivity of LC-MS/MS analysis: implication for proteomics experiments‘, Journal of Proteomics, published online November 14, 2012. http://dx.doi.org/10.1016/j.jprot.2012.11.005