Low-pg/mL quantitation of leucine-rich repeat kinase 2 (LRRK2) protein in human cerebrospinal fluid (CSF)


Streamlined and sensitive workflow using the M5 MicroLC system and the SCIEX 7500 system

Dylan Bennett1 , Arash Rassoulpour1 , Ebru Selen2 , Rahul Baghla2 and Eshani Nandita2
1Neuron23, USA; 2SCIEX, USA

Abstract


This technical note demonstrates a sensitive method for the quantitation of LRRK2 in human CSF using triple quadrupole mass spectrometry. The method uses a Stable Isotope Standard Capture by Anti-peptide Antibody (SISCAPA) sample preparation methodology. A lower limit of quantitation (LLOQ) of 10 pg/mL was achieved in human CSF (Figure 1).

Introduction


Parkinson's disease is the second-most common neurodegenerative disorder, affecting millions worldwide. It is characterized by symptoms such as tremors, stiffness, slowed movements and impaired balance.1-3 While the exact cause of Parkinson's remains unknown, both genetic and environmental factors are believed to contribute to its development. Research has shown mutations in the gene encoding LRRK2 to be one of the most common causes of the disease. The ability to detect and quantify LRRK2 protein in human CSF will be a crucial companion diagnostic to a therapy that might modulate disease state and protein levels.

This technical note presents a reliable and highly sensitive workflow to support the routine quantitative analysis of LRRK2 protein using the SCIEX 7500 system coupled with the M5 MicroLC system.

Figure 1. Representative extracted ion chromatograms (XICs) for LRRK2 in human plasma. The left panel displays the XIC at the LLOQ (25 pg/mL) with analytical flow LC conditions. The middle panel depicts the XIC at the LLOQ (10 pg/mL) level with the microflow LC experiment, indicating a significant 2.5x improvement in LLOQ.The right panel illustrates the XIC at 25 pg/mL for the microflow LC conditions, which shows a 5x gain in S/N over the analytical flow experiment.

Key features for the analysis of LRRK2 protein using the SCIEX 7500 system coupled with the M5 MicroLC system
 

  • Low pg/mL level of quantitation: Achieve 10 pg/mL LLOQ for quantitation of LRRK2 protein in human CSF

  • Enhanced sensitivity: Achieve a 2.5-fold improvement in LLOQ and a 5-fold improvement in signal-to-noise (S/N) using the M5 MicroLC system compared to conventional analytical flow conditions

  • Lower solvent consumption: The trap-and-elute workflow uses up to 50x less solvent than the analytical flow method, resulting in significant cost savings, waste reduction and a more sustainable solution

  • Robust analytical performance: Achieve accurate quantitative performance with %CV <10% at all concentration levels across a linear dynamic range (LDR) of 3 orders of magnitude

  • Streamlined data management: Data acquisition and processing are simplified with SCIEX OS software, a 21 CFR Part 11-compliant platform
     

Methods


Sample preparation: Calibration standards were prepared at concentrations ranging from 10–10000 pg/mL by serially diluting recombinant LRRK2 protein (rLRRK2) using 0.1% BSA in 1X PBS as a surrogate matrix. A total of 1 mL of standard samples, human CSF samples and blanks were first treated with radioimmunoprecipitation assay (RIPA) buffer. Digestion was performed using trypsin for 90 minutes at 40°C with shaking. After digestion, SIL synthetic peptide was spiked into the samples to serve as an internal standard (IS).

Immunoaffinity extraction was performed on the digests using anti-peptide antibody-coated magnetic beads to capture the signature peptide, KAEEKAEEGDLLVNPDQPR (KAEE peptide), used for quantitative analysis. Samples and beads were incubated at 4°C for 90 minutes on an end-over-end rotating mixer. After several washing steps, the KAEE peptide was eluted from the beads before LC-MS/MS analysis.1

Mass spectrometry: Samples were analyzed using the SCIEX 7500 system in MRM mode. The system was controlled by SCIEX OS software. The optimized MS parameters are listed in Table 1.

Table 1. MS and source parameters.

Chromatography: Chromatographic separation was performed on the M5 MicroLC system used in the trap-and-elute mode. The 20 µL sample was loaded onto the Phenomenex Luna C18 (0.3 mm x 10 mm) micro trap for 1 minute at a 40 µL/mL loading flow rate. Analytical separation was performed at a 5 µL/mL flow rate using a Kinetex XB-C18 (2.6 µm, 0.3 mm x 50 mm, 100 Å) microflow LC column. Samples were run with a 10-minute gradient. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was 0.1% (v/v) formic acid in acetonitrile. The LC column was operated at 35°C. Gradient conditions are summarized in Table 2.

Table 2. LC conditions.

Data processing: Data processing was performed using SCIEX OS software, version 3.1. Peaks were automatically integrated using the MQ4 algorithm with a weighting of 1/x2.

Quantitative performance


A calibration curve was analyzed for concentrations ranging from 10–10000 pg/mL. To evaluate reproducibility, each concentration was analyzed in triplicate.

The LLOQ achieved for LRRK2 protein in human CSF was accurately measured at 10 pg/mL. No interferences were observed in the blank matrix (Figure 2).

Figure 2. Representative XICs of the matrix blank and LLOQ of the LRRK2 protein spiked at 10 pg/mL.

Linearity was achieved across a range of concentrations from 10–10000 pg/mL with a correlation of determination (r2) of 0.996 for LRRK2 protein in the CSF surrogate matrix (Figure 3). A linear dynamic range (LDR) of 3 orders of magnitude was reached.

Figure 3. Calibration curve for the quantitation of LRRK2 protein. Linearity was established between 10 pg/mL and 10000 pg/mL, generating an LDR of 3 orders of magnitude with an r2 of 0.996.

Analytical performance was evaluated based on the criteria that the accuracy of the calculated mean should be between 80% and 120% at the LOQ and between 85% and 115% at the higher concentrations. In addition, the %CV of the calculated mean of the concentration should be <20% at the LLOQ and <15% at all higher concentrations.

The assay accuracy was within ±4% of the actual concentration and the %CV was <10%. Calculated percent accuracy and %CV values were within the acceptance criteria at each concentration level (Figure 4).

Figure 4. Quantitative performance for LRRK2 protein analysis. Reproducibility and accuracy results were determined from the calibration curve standards across 3 replicates at each concentration. Statistical results were summarized using the Analytics module in SCIEX OS software.

Matrix equivalency


Since experiments were performed in the surrogate matrix, a matrix equivalency experiment was carried out to ensure quantitative performance aligns with the human CSF samples. Pooled human CSF samples were analyzed for their endogenous LRRK2 concentration and spiked with the known concentrations of LRRK2 protein used for calibration standards. The calculated concentrations for all spiked samples were within 10% of the theoretical post-spike concentrations. Results meet regulatory requirements and suggest good matrix equivalency and an absence of matrix effects (Table 3).

Table 3. Investigation of matrix equivalency.

Additional human CSF samples were analyzed to measure endogenous LRRK2 protein levels with concentrations ranging from 7.4 pg/mL (below the quantitation limit) to 86.9 pg/mL (Figure 5).

Figure 5. Representative XICs from CSF samples. The left panel shows unspiked human CSF samples, while the right panel displays human CSF samples spiked with 50 pg/mL of LRRK2 protein.

Enhanced sensitivity with the M5 MicroLC system


Matrix-extracted samples were also analyzed using analytical flow conditions at 0.4 mL/min using the Kinetex XB-C18 column (2.1 mm x 100 mm, 1.7 µm, 100 Å) with the same mobile phase conditions described in Table 2. Data were compared between the 2 systems. The results showed a 2.5- fold improvement in LLOQ and a 5-fold improvement in S/N using the M5 MicroLC system compared to conventional analytical flow conditions (Figure 6).

Figure 6. Representative XICs from analytical flow and microflow experiments. The left panel displays the XIC of LLOQ at 25 pg/mL using analytical flow conditions. The right panel shows the XIC of LLOQ at 25 pg/mL with microflow conditions, demonstrating a 2.5x improvement in LLOQ

Compliance-ready SCIEX OS software


SCIEX OS software is a closed system and requires records and signatures to be stored electronically, meeting the regulations outlined in 21 CFR Part 11. SCIEX OS software can open raw data files from any visible storage location within a closed network by using designated processing workstations. Figure 7 illustrates the features of the SCIEX OS software for monitoring the audit trail, performing acquisition, processing data and configuring user access. The audit trail feature enables users to monitor high-risk events and evaluate the data integrity. The Central Administrator Console (CAC) feature allows users to centralize acquisition and processing in a single platform to enable higher efficiency for multi-instrument laboratories, whether for meeting regulated or non-regulated compliance standards. Using the configuration module, users can assign roles and access to the administrator, method developer, analyst and reviewer.

Figure 7. Features on the SCIEX OS software for monitoring user access and evaluating the audit trail. The audit trail view allows users to filter for high-risk events easily and enables data integrity features to meet compliance requirements. The software features a Central Administrator Console (CAC) to monitor acquisition and processing across all systems in a centralized manner. The CAC feature supports both regulated and non-regulated compliance standards. The configuration module enables users to easily set up roles and levels of access for the administrator, method developer, analyst and reviewer roles.

Conclusion
 

  • An LLOQ of 10 pg/mL was achieved to quantify LRRK2 protein in human CSF samples using LRRK2-SISCAPA methodology

  • Good linearity was achieved across concentrations ranging from 10–10000 pg/mL with an r2 of 0.996 and an LDR spanning 3 orders of magnitude

  • Compared to conventional analytical flow conditions, a 2.5- fold improvement in LLOQ and a 5-fold improvement in S/N were achieved using the M5 MicroLC system. The improvement in sensitivity could allow for a more robust assay in which smaller volumes can be injected with improved limits of quantitation.

  • Accurate and highly reproducible quantitative performance was demonstrated at all concentration levels with a %CV <10%

  • Matrix equivalency between the surrogate and authentic matrices was achieved with no significant matrix effects observed

  • Up to 50x lower solvent consumption was achieved with the microflow method using a trap-and-elute setup compared to an analytical flow method. Minimizing LC-MS-grade solvent consumption increases cost savings, reduces waste generation and promotes an eco-friendly option.

  • The assay exhibited the ability to routinely quantify low levels of LRRK2 protein with a 10-minute runtime, enabling bioanalytical labs to deliver high-quality data with good throughput
     
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