Significant gains in quantitative sensitivity using microflow chromatography

Methods

Sample preparation: Rat plasma was crashed with acetonitrile (2:1, acetonitrile/plasma). The sample was mixed and then centrifuged at 16,000 rpm for 6 minutes to pelletize the protein. The supernatant was collected and diluted 5 fold with water to use as the matrix.

A panel of small molecules was dissolved in methanol and mixed together to make a 10 μg/mL stock solution. This solution was diluted to create a concentration curve, which was then diluted 10x with crashed plasma to create the final solutions with matrix (covering the concentration range of 0.001 to 2500 pg/mL).

PepCalMix (a mixture of 20 different tryptic peptides, SCIEX) was diluted in 5% acetic acid, 2% acetonitrile in 0.1% formic acid to create a 10 fmol/μL stock solution. This was then diluted into simple matrix to create a standard concentration curve.

Chromatography: Separations were performed using a NanoLC™ 425 System (SCIEX) operating in low microflow range and controlled by contact closure.6 The flow rate was 3 μL/min. The gradients used are outlined in Table 1.

Mass spectrometry: Small molecules and peptides were analyzed under very similar microflow LC conditions on both the SCIEX 7500 System and the QTRAP 6500+ System. Source conditions and some key instrument specific parameters were optimized between platforms.

Data processing: Data was processed using SCIEX OS Software 2.0 using both the Explorer and Analytics modules.

Small molecule quantification

A suite of small molecule compounds were used to benchmark the sensitivity gains between the SCIEX Triple Quad 7500 System – QTRAP Ready and the QTRAP 6500+ System in the microflow chromatography regime. Three systems of each type were used to ensure a robust sensitivity comparison was obtained. Source and compound dependent parameters were optimized on each system to ensure best sensitivity on each instrument. Collision energies between the two platforms were optimized to the same values for each compound. Source settings did vary between platforms due to the differences between the OptiFlow Pro Ion Source and the IonDrive™ Turbo V Source. Calibration curves were run across a broad range of concentrations in protein-precipitated rat plasma, to compare the observed peak areas as well as determine the lower limits of quantification for the range of compounds. Lower limits of quantification were compared for these small molecules in crashed rat plasma between the SCIEX 7500 System and the QTRAP 6500+ System (Figure 4), an average improvement in LLOQ of 2.9 was observed.

Peptide quantification 

Similar experiments were performed on a set of tryptic peptides in simple matrix to determine typical sensitivity gains for peptides at microflow rates between the SCIEX Triple Quad 7500 System – QTRAP Ready and the QTRAP 6500+ System. Again concentration curves across a broad range of concentrations were run to compare both the peak areas (Figure 6) and the LLOQs (Figure 5). An average improvement for the SCIEX 7500 System over the QTRAP 6500+ System was a 4.6 fold lower LLOQ across the 15 peptides monitored.

Conclusions

The quantification workflows for peptides and small molecules were explored on the SCIEX Triple Quad 7500 System – QTRAP Ready, to characterize the sensitivity improvements over the previous generation of instruments. Calibration curves were run on three SCIEX 7500 Systems and three QTRAP 6500+ Systems, both with microflow LC, and the LLOQs and peak areas were compared. Average peak area gains of about 7.5 were observed across the set of compounds analyzed (Figure 1). LLOQ improvements of 2.9 for small molecules and 4.6 for peptides were observed for the SCIEX 7500 System. In all, this work demonstrates that the improvements in the SCIEX 7500 System also provide sensitivity gains in the microflow regime.

References

1. Ultra-sensitive quantification of trastuzumab emtansine in mouse plasma using trap-elute microLC-MS method. SCIEX technical note RUO-MKT-02-8288-A.
2.  Microflow chromatography: Bridging the sensitivity gap for quantitation of a pyrrolobenzodiazepine dimer. SCIEX technical note RUO-MKT-02-9512-A.
3. Microflow SWATH Acquisition for industrialized quantitative proteomics. SCIEX Technical Note RUO-MKT-02-3637-A.
4. Single source solution for low flow chromatography. SCIEX technical note RUO-MKT-02-9701-A.
5. Enabling new levels of quantification. SCIEX technical note RUO-MKT-02-11886-A.
6. Controlling SCIEX microflow LC Systems using contact closure in SCIEX OS Software. SCIEX community post RUO-MKT-18-11908-A.