The spleen tissue samples were thawed on ice, diced into small pieces and then homogenized with a Storm Pro bullet blender (Next Advance).
Upon detection of 4 out of 6 product ions from the SIL, the light peptides were acquired via a data-dependent MS2 scan with an isolation offset dependent on the terminal amino acid and charge state at a resolution of 60,000 with an AGC setting of 1 x 106, a MIT of 116 msec, and a NCE of 30. Upon detection of the SIL peptide precursors in the MS1 scan, a data-dependent MS2 scan of the heavy peptides was acquired at a resolution of 7,500 with an AGC setting of 1 x 10^6, a MIT of 10 msec, and a NCE of 30. An MS1 scan from 300 m/z to 1000 m/z was performed at 120,000 resolution with an AGC target of 1 x 10^6 and a MIT of 50 msec. These levels were then adjusted empirically to minimize triggering on spurious peaks. The MS1 intensity thresholds for acquisition of the SIL peptides were initially set at 0.005% of the max signal intensity for the SILs obtained from a survey run. The MS2 scans were acquired at a resolution of 30,000 with an AGC setting of 1 x 10^5, a MIT of 80 msec, and a NCE of 30. A mixture of only SIL peptides in similar biological matrix was used to determine the concentrations and intensity threshold settings needed for optimal triggering. Finally, IS-PRM was also performed on an Exploris 480 using the Thermo Scientific SureQuant methodology.
The MS2 scans were acquired at a resolution of 30,000 with an automatic gain control (AGC) setting of 1 x 10^5, a maximum injection time (MIT) of 80 msec, and a normalized HCD collision energy (NCE) of 30. A scheduling window of 1 min was also used for each peptide monitored. Alternatively, PRM analyses were performed on an Exploris 480 quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific). The MRM method utilized a 1 min scheduling window for each peptide monitored and a cycle time of 1 sec. Collision energy optimization and fragment ion selection (2-4 transitions per peptide) were done by analyzing recombinant kinase digests with Skyline. MRM analyses were performed on a TSQ Altis triple quadrupole mass spectrometer (Thermo Fisher Scientific). After elution from the column, the peptides were introduced to the mass spectrometers with an EASYSpray ion source with an ionization voltage of 1.7 kV. The separation was done using a gradient of solvents A (0.1% formic acid, 2% acetonitrile in water) and B (0.1% formic acid, 10% water, 90% acetonitrile) as follows: 1% B for 2 min, then a gradient of 1-45% B over 30 min, then an increase to 90% B for 6 min. After injection of 2 µL per sample, the peptides were separated on an EASYSpray C18 reverse phase column (75 µm x 25 cm) heated to 50° C with a flow rate of 300 nL/min.
Understanding these differences could help rationalize the findings of preclinical studies and have major implications for the selection of these animals as models in kinase drug development.Īn UltiMate 3000 UPLC system (Dionex) with the same parameters and configuration across methods and instruments was used to eliminate possible sources of variance. IS-PRM provided the highest number of kinase identifications, and the results indicate that while this initial set of kinases exhibits high correlation between species for this tissue type, discreet species-specific differences do exist, especially within the cyclin-dependent kinase (CDK) family. These methods were then used to profile inter-species kinome variability in spleen with three of the current techniques used in targeted proteomics (MRM, PRM and IS-PRM). We report the development of a targeted mass spectrometry-based assay capable of monitoring >50 different kinases using peptides conserved in humans and the key preclinical species used in drug development (mouse, rat, dog and cynomolgus monkey). Little is currently known about how kinase expression can vary between species, and tools to quantitatively measure such differences are lacking. This problem may be exacerbated by the fact that these studies might fail to consider the basic physiological differences which could exist between human patients and animal models. Early attrition of drug candidates, including kinase inhibitors, often occurs due to issues that arise during preclinical safety and efficacy evaluation.