Differential mobility separation mass spectrometry for quantitation of large peptides in biological matrices
Using SelexION® Technology coupled with a QTRAP® 5500 System
Derek T. Wachtel, Sanjeev T. Forsyth & Robert W. Busby
Ironwood Pharmaceuticals, Cambridge, MA
The ability to quantify large peptides in biological matrix with adequate selectivity and sensitivity depends on several factors: 1) peptides are multiply charged, 2) peptides have varied fragmentation efficiency due size and structure, and 3) different matrices can cause varied background signal and interferences. Tryptic digestion can reduce the size of the peptide and improve fragmentation but introduces an additional step, which is undesirable in a high-throughput environment.
Detection limits for MRM acquisition can be heavily affected by the fragmentation characteristics of the peptide. Sensitivity is reduced if the peptide does not fragment or if it fragments too extensively (spreading ion current across many product ions). Cyclic peptides are especially difficult as they tend to have poor MS/MS efficiency. For these cases, high sensitivity can sometimes be achieved through monitoring the intact peptide in Single Ion Monitoring (SIM) mode. Methods based on SIM are not routinely utilized because they can have reduced selectivity and high background levels.
Differential mobility separation (DMS) mass spectrometry adds an additional level of selectivity to LC-MS/MS providing gas phase separation of isobaric species and co-eluting interferences to reduce background noise. Here, the utility of a SIM workflow combined with DMS for specificity was investigated for quantifying large therapeutic peptides in a protein precipitated plasma matrix.
Sample preparation: To extract the peptides from plasma, an aliquot of 100 µL of each standard was mixed with 400 µL of 5% formic acid in acetonitrile (with 150 ng/mL peptide). This was vortexed for 10 mins, then centrifuged at room temperature for 10 mins (21000 x g). The supernatant was transferred (425 µL) to a clean 96 well plate and evaporated to dryness at 50 ºC. To reconstitute the sample, 30 µL of acetonitrile was added and the sample was placed on the shaker for 15 mins. An additional 20 µL of 2.5% formic acid in water was added and the sample was shaken for an additional 15 mins.
HPLC conditions: The samples were analyzed using the Acquity UPLC system (Waters). The peptides were directly loaded onto a 2.1 x 50 mm column (Thermo Hypersil Gold, 1.9 µm). An elution gradient of 5-95% acetonitrile (0.1% formic acid) over 3 min was used with a flow rate of 800 µL/min.
MS/MS conditions: A QTRAP 5500 System equipped with SelexION Technology and a Turbo V™ Ion Source was used. DMS parameters were optimized for each peptide of interest to maximize signal intensity, the following parameters were used (DMS temp → low, Modifier → none, Separation Voltage → 3800, Compensation Voltage → 15.5, DMO Offset → -5.5, DMS Resolution → off). Analysis is done using a single ion monitoring (SIM) acquisition strategy.
Single ion monitoring (SIM) is a variation of an MRM experiment where the parent ion is monitored in Q1 and Q3 without fragmentation in Q2. For large peptides, SIM methods can provide larger signal without the need for extensive compound optimization.
Data processing: Results were analyzed using Analyst® Software 1.5 Quantification tools.
High-Throughput Peptide Quantitation
It is undesirable in a high-throughput setting to optimize the extraction method and chromatographic conditions for every test compound being evaluated. A simple extraction method along with high throughput chromatographic conditions were developed and optimized for selectivity and sensitivity for a diverse set of peptides (~30 amino acids, 4 kDa) in multiple lots of rat plasma for analysis by LC-MS/MS.
In addition, generic MS acquisition methods are attractive but often limited by background noise and deliver reduced sensitivity, limiting the ability to generate a suitable pharmacokinetic (PK) profile for a peptide. MRM methods are the gold standard for high sensitivity quantification but sometimes on large peptides, the fragmentation properties of the peptide can limit the ultimate sensitivity achieved (Figure 2).
Monitoring the intact form of the peptide using SIM can provide better sensitivity (Figure 3, top) but have much higher background levels which can also impact detection limits.
Combining the higher intensity MS method with differential mobility separation can provide the added selectivity needed to improve detection limits. Figure 3 (bottom) shows the improvement in S/N observed vs using SIM alone, a significant decrease in background was observed.
Assay Performance using DMS with SIM
The lower limit of quantification (LLOQ) for the DMS + SIM acquisition strategy was 4 ng/mL (Figure 4, top) while conventional MRM was limited to 125 ng/mL (Figure 2), an ~30 fold improvement in detection limit. The statistics for the peaks observed at 4 ng/mL are shown in the table (Figure 4), good reproducibility and accuracy was achieved.