Quantification of Amyloid Beta Peptides in Human ...

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Introduction Amyloid peptides (Abeta; Aβ) result from cleavage of amyloid precursor protein (APP) by beta and gamma secretase. The 42-amino acid variant, amyloid β peptide 1–42 is a widely accepted key biomarker for Alzheimer's disease (AD) together with the total tau (T-tau) and phosphorylated tau (P-tau) protein. Diagnostic accuracy of the Aβ 1–42 /Aβ 1–40 ratio in cerebrospinal uid (CSF) compared to the concentration of Aβ 1–42 alone was found to be even more signicant. Currently these peptides are analyzed routinely in CSF by immunoassays, but in recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been investigated for the quantication of Aβ1-38, Aβ1-40, and Aβ1–42. [Blennow 2010, Lewczuk 2015, Lame 2011] Authors Antje Meyer¹, Christoph Siethoff², Filip Sucharski², Matthias Orth² ¹ Probiodrug, Halle, Germany ² Swiss BioQuant, Reinach, Switzerland Figure 1: Solution structure of the Alzheimer's disease amyloid beta-peptide (1-42) Quantication of Amyloid Beta Peptides in Human Cerebrospinal Fluid using Micro HPLC-HRMS Application Note

Transcript of Quantification of Amyloid Beta Peptides in Human ...

Page 1: Quantification of Amyloid Beta Peptides in Human ...

Introduction

Amyloid peptides (Abeta; Aβ) result from cleavage of amyloid precursor protein (APP) by beta and gamma secretase. The 42-amino acid variant, amyloid β peptide 1–42 is a widely accepted key biomarker for Alzheimer's disease (AD) together with the total tau (T-tau) and phosphorylated tau (P-tau) protein. Diagnostic accuracy of the Aβ 1–42 /Aβ 1–40 ratio in cerebrospinal fluid (CSF) compared to the concentration of Aβ 1–42 alone was found to be even more significant. Currently these peptides are analyzed routinely in CSF by immunoassays, but in recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been investigated for the quantification of Aβ1-38, Aβ1-40, and Aβ1–42.[Blennow 2010, Lewczuk 2015, Lame 2011]

Authors

Antje Meyer¹, ChristophSiethoff², Filip Sucharski², Matthias Orth²

¹ Probiodrug, Halle, Germany² Swiss BioQuant, Reinach, Switzerland

Figure 1: Solution structure of the Alzheimer's disease amyloid beta-peptide (1-42)

Quantification of Amyloid Beta Peptides in Human Cerebrospinal Fluid using Micro HPLC-HRMSApplication Note

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Instrumentation

HPLC system:Analytical Pump:Zirconium Ultra 410F (Prolab, Reinach, Switzerland)

PAL HTS autosampler (CTC Analytics, Zwingen, Switzerland)

Dilution and loading pumps:Binary pumps 1200 (Agilent Technologies, Santa Clara, USA)

Trapping column: Gemini C18, 5 µm, Zircotrap, 1 mm ID x 8 mm (Phenomenex, Prolab)Flow rate: 250 µL/min (50 µL/min Sample flow plus 200 µL/min dilution flow)

Analytical column: BEH C18, 300 A, 1.7 µm, 0.3 mm ID x 150 mm (Waters),50°CPhase A: water containing 0.2 % ammoniaPhase B: acetonitrile containing 0.2 % ammoniaFlow rate: 4 to 3 µL/min

MS system:LTQ Orbitrap XL (Thermo, Bremen, Germany)Prolab µSource and Column oven

Scan mode:Full scan from m/z 900 to 1200Resolution at 15000Positive ion mode

Figure 3: Instrumentation Setup with Load and Dilution Pumps, Autosampler, Analytical Pump, and Mass Spectrometer

AC

Analytical Pump4µl/min Bin. Gradient

Dilution Pump200 µl/min H2O

Load Pump50 µl/min H2O

Figure 4: Flow schematic - loading sample into sample loop

AC

Analytical Pump4µl/min Bin. Gradient

Dilution Pump200 µl/min H2O

Load Pump50 µl/min H2O

Figure 5: Flow schematic - transfer sample to trapping column

AC

Analytical Pump4µl/min Bin. Gradient

Dilution Pump200 µl/min H2O

Load Pump50 µl/min H2O

Figure 6: Flow schematic - switch trapping column into analytical flow pathFigure 2: LC gradient/flow profile

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Figure 7: Calibration Curve. Concentration range: 200 pg/mL to 20.0 ng/mL

Figure 12: Reproducibility - 8 sequential runs at LLOQ

Figure 9: LLOQ - Chromatogram CSF sample - IP enrichmentFigure 8: LLOQ - Chromatogram (detail)

Figure 11: LLOQ - Spectrum zoomed in at Abeta 1-40

Methodology

The CSF sample was prepared by immuno-precipitation enrichment, after which it contained 50% organic (ACN). 50 µL of the prepared sample were injected with the autosampler (figure 4). After switching the injector valve, the sample was transferred to the trapping column during 3 minutes using 50 µL/min flow through the injection loop, and an additional 200 µL/min H2O added after the loop to dilute the sample for trapping (figure 5).The trapping column was then switched into the analytical LC flow path (figure 6) and extracted at an initial 4 µL/min flow rate, which was reduced to 3 µL/min after 8 minutes in order to broaden the peaks around the retention time of interest (figure 2).

Figure 10: LLOQ - Spectrum at 9.71 min.

Results and Conclusions

The linearity at the dynamic range of 100 is R² = 0.9917 (figure 7). The sensitivity at the chosen LLOQ of 200 pg/mL suggests a feasible detection limit below 20 pg/mL (figure 8). Reproducibility of both the area and the retention times (figure 12) is excellent.

The use of the combination of microflow HPLC and high resolution mass spectrometry for quantitation was demonstrated by the analysis of beta amyloid peptides 1-40 and 1-42 in human cerebrospinal fluid. Sensitivity in the low pg/mL range could be achieved by reducing the flow rate and the inner diameter of the HPLC column, but still without reducing the injection volume to get the real advantage of microflow HPLC-MS with respect to sensitivity gain. Quantitation by high resolution mass spectrometry was precise and robust, which allows us to use this method for a biomarker clinical study.

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References

Blennow K, Hampel H, Weiner M, Zetterberg H. Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nat Rev Neurol. 2010 Mar;6(3):131-44.

Lewczuk P, Lelental N, Spitzer P, Maler JM, Kornhuber J. Amyloid-β 42/40 cerebrospinal fluid concentration ratio in the diagnostics of Alzheimer's disease: validation of two novel assays. J Alzheimers Dis. 2015;43(1):183-91.

Lame ME, Chambers EE, Blatnik M. Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry. Anal Biochem. 2011

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