Hello, I am trying to quantify some very low abundance peptides and need to squeeze every last bit of sensitivity out of my instrument (Waters Xevo tq-s). Is it possible to collect SRM data and then combine the signal from multiple product ions? Would this actually lead to a useful increase in sensitivity? Thank you for your help
Yes, it is possible to collect SRM (Selective Reaction Monitoring) data and then combine the signal from multiple product ions to increase sensitivity in quantitative analysisCombining the signal from multiple product ions can improve the sensitivity of your analysis by increasing the total ion count (TIC) and reducing the contribution of background noise. This approach is often referred to as Multiple Reaction Monitoring (MRM) or MRMCombining the signal from multiple product ions can potentially improve sensitivity as it increases the overall signal intensity. This approach is commonly known as multiple reaction monitoring (MRM) or scheduled MRM.
In traditional SRM, a single precursor ion is selected and fragmented into one or a few product ions. The specific transitions monitored are typically chosen based on the peptide sequence and the peptide fragmentation pattern. However, in cases of very low abundance peptides, the signal intensity from a single transition might be too weak for accurate quantification.
To enhance sensitivity, you can employ a strategy called "multiplexing" or "multiplexed MRM." In this approach, multiple product ions from the same precursor ion are monitored simultaneously. By summing the signal from these multiple transitions, the overall signal intensity is increased, resulting in improved sensitivity.
To implement multiplexed MRM, you need to carefully select transitions that provide sufficient specificity and sensitivity while avoiding interferences. It is essential to consider the selectivity of the transitions, signal-to-noise ratio, and potential interference from co-eluting species.
Additionally, optimizing your LC-MS/MS parameters such as the collision energy, dwell time, and precursor ion selection can also help in improving sensitivity.
By combining the signal from multiple product ions in multiplexed MRM, you may achieve a useful increase in sensitivity, making it beneficial for quantifying low-abundance peptides.
Combining multiple ions from multiple ion reaction mechanisms is effectively the same as quantifying based upon total ion chromatogram mass spectra. That being said, in an MS/MS application, careful control of both of the MS steps with respect to fragmentation and ionization will be necessary to avoid significant variability in the results. There is also the question of whether your software will allow you to do this.
As always use caution in interpreting the data especially with respect to non-zero intercepts in the sample response curves.
As noted by Taban, using 'MRM' mode accomplishes this and maintains selectivity (which TIC may not). Additionally, time should be spend OPTIMIZING the HPLC analysis method, detector settings (gas flow, temp, voltages etc), reducing the use of any additives which may contribute to higher background noise signals, cleaning and servicing the instruments. *Improvements to S/N translate to improvements to sensitivity. Never rely on "area" or "ion" counts alone. You must measure S/N for each peak (sample) to determine detection levels.
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