Everything is perfect. You’re on the beach, sunlight is ideal, just a calm breeze to gently move the hair and you snap the photo using your mobile phone. You immediately look at the picture in gleeful anticipation, only to find someone walking in the background. The picture is ruined! You know that was a one-in-a-million shot, and try as you might, none of the other 50 shots match that one golden image! At least until now. Using modern mobile phones and accompanying software, you can erase the background images from your photo to restore it to a Rembrandt with tremendous ease.
The essence of photobombing holds true for targeted protein quantitation. You know the protein of interest, have identified the key peptide containing the active site and its optimal product ions to establish the most selective, sensitive, and specific SRM transitions using the ideal chromatographic separation maintaining the desired throughput. You run the method through the initial tests against a representative matrix for verification.
Everything looks fine until you begin acquiring data on real donor samples, and 30 percent of the samples show background interference, limiting your quantitative sensitivity.
Generally, this requires you to extend the chromatographic gradient to improve peak capacity and separate the background interference from the target peptide elution. More drastic measures to maintain the same sample throughput may include modifying sample preparation for targeted extraction, requiring much greater resources in time and money.
Professor Jun Qu, Qingqing Shen and colleagues at the State University of New York at Buffalo have developed a more efficient and effective method to erase unwanted background interference delivering desired limits of quantitation (LOQ) faster and easier. They utilize LC-FAIMS-MS/MS workflows to minimize background interference for their biotherapeutic PK and drug target studies.
FAIMS Pro Duo interface introduced
Introducing the Thermo Scientific™ FAIMS Pro Duo interface into their workflow enables the team to apply their novel differential compensation voltage (dCV) approach and quickly determine the optimal CV setting, improving the signal-to-noise ratio (S/N) needed to achieve the desired LOQ measurements.
The FAIMS Pro Duo interface utilizes differential ion mobility as an additional selectivity filter on the millisecond timescale that is orthogonal to separation conditions of reverse phase (RP) chromatography and mass spectrometry, resulting in a
tailored beam transmission through interface and into the mass spectrometer as shown in Figure 1. FAIMS separation also operates on the same millisecond timescale as SRM transitions without affecting the cycle time. The voltage fields can be “tuned” for different peptides using a compensation voltage (CV) that can be adjusted between -100 and 100 V to erase unwanted ions. With the recent introduction of the Thermo Scientific™ TSQ Altis Plus and TSQ Quantis™ Plus triple quadrupole mass spectrometers, software tools enable online CV analysis to simplify the dCV process. The resulting data can be processed and viewed in Thermo Scientific™ FreeStyle™ software.
Using the differential CV (dCV) approach
The CV setting for optimal transmission per targeted peptide is determined by analyzing the matrix with and without the spiked biotherapeutic. Figure 2 overlays the measured signal for both samples as a function of CV settings to determine the window of opportunity, that is, the CV setting providing the greatest difference in measured response SRM transitions when the target peptide is in the sample matrix. This unique approach is the differential CV (dCV) approach. As with any quantitative experiment, maximizing S/N to extend the measured LOQ is the ultimate goal, not simply boosting target peptide signal, which may also boost the photobombing background ions.
Comparing LOQ determination
To demonstrate, Professor Qu and his team performed a series of experiments comparing LOQ determination for a set of endogenous biomarkers in human plasma. The SRM response measured for the surrogate protein biomarkers with and without FAIMS selectivity following dCV determination as well as evaluating the dCV response was used to evaluate the benefit of the new workflow. Figure 3 shows the evaluation for targeted peptide from Lipocalin 2 in pooled human plasma. Without FAIMS selectivity, we have, in effect, photobombing from background interference delivering a poor S/N. Removing the background with the optimal CV setting shows a 45-fold improvement in S/N that is picture perfect. The dCV traces showed background interference having a similar CV maximum as that for the spiked peptide at -43 V, but the window of opportunity was determined to be -55 V. While the “optimal filter” reduced the measured SRM intensity by 30 percent, reaching the target LOQ level in time to enjoy the beach is always welcomed.
Of course, photobombing may be more severe than simply someone walking in the background. Occasionally, the picture appears to be unsalvageable. Stubborn interference can also be encountered when analyzing biological samples. There are often instances where background peptides have very similar sequences to that of the targeted peptide. The peptide sequences govern the physicochemical properties of peptides, such as the molecular weight, ionized charge state, and the dipole moment defining the ion mobility in the high- and low-electric fields.
These situations may preclude FAIMS selectivity from removing the background when operated under standard conditions, regardless of CV setting. Figure 4 shows the comparative SRM response for the surrogate peptide biomarker for Dipeptidyl-peptidase 4 in human plasma.
The data was acquired with and without FAIMS selectivity using standard operating parameters. FAIMS selectivity does clean up the background interference displayed in the retention time window, boosting S/N. Evaluation of the targeted SRM peak shape with FAIMS selectivity, however, shows shoulders indicating co-eluting background interference that may mask the true signal.
Evaluation of the dCV analysis using standard FAIMS Pro Duo settings (Figure 5) show similar SRM intensity across the range of CV settings of the blank matrix and spiked standard, resulting in no windows of opportunity to dramatically improve the S/N ratio.
One can, however, change the temperature of the neutral carrier gas to modify the ion mobility characteristics within the FAIMS Pro Duo interface. Operating the FAIMS Pro Duo interface in “high resolution” mode (Figure 6) modifies the dCV profiles of measured SRM response for the background matrix and spiked samples.
Note that operating the FAIMS Pro Duo interface in the high-resolution setting can shift the identified CV maximum as identified using standard operating conditions.
The results of using high-resolution settings for the FAIMS Pro Duo interface create a window of opportunity where none existed before, resulting in a S/N of 190 and a “publishable” SRM trace (Figure 7).
The challenge of targeted peptide quantitation
Targeted peptide quantitation in the presence of biological matrices is challenging. Not all peptides face the same probability of background interference, thus researchers need tools to handle those situations when a problem appears for two of the 10 peptides easily and rapidly, while not adversely affecting the other eight peptides.
Implementation of the FAIMS Pro Duo interface into the LC-MS/MS workflow provides many advantages including the ability to erase photobombing by unwanted ions. Installation of the FAIMS Pro Duo interface is very quick and works for experimental methods covering a wide range of chromatographic flow rates from 100 nL/min to 1000 µL/min using a variety of ESI sources (Figure 8).
The new TSQ and TSQ Plus MS software enables online CV optimization to perform dCV measurements as a function of the chromatographic separation to provide realistic improvements to the overall method where all targeted peptides can benefit as shown to the right. The CV switching speed is also in line with fast SRM acquisition speeds to facilitate multiple CV settings within a chromatographic time window. Lastly, the FAIMS Pro Duo interface electrode assembly provides a barrier to direct line-of-sight for ions to block neutrals and salts from entering the mass spectrometer, increasing the overall method robustness.
For more information on Prof. Jun Qu’s work: https://www.acsu.buffalo.edu/~junqu/members_junqu.html
For more information about the key components of the workflow: