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Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry.

Bros P, Vialaret J, Barthelemy N, Delatour V, Gabelle A, Lehmann S, Hirtz C - Front Neurosci (2015)

Bottom Line: Quantification was performed using isotopically labeled (15)N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs).Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides.This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biochimie et de Protéomique Clinique - Institut de Médecine Régénérative et Biothérapies, Centre Hospitalier Universitaire de Montpellier Montpellier, France ; Laboratoire National de Métrologie et d'Essais (LNE) Paris, France.

ABSTRACT
Tau protein concentration in cerebrospinal fluid (CSF) is currently used as a sensitive and specific biomarker for Alzheimer's disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS) to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid (PCA) followed by micro solid phase extraction (μSPE). We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM) on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labeled (15)N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs). Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

No MeSH data available.


Related in: MedlinePlus

Calibration curves of the 7 tau peptides.
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Figure 3: Calibration curves of the 7 tau peptides.

Mentions: Between successive LC-MS/MS analysis, no memory effect phenomenon was observed. The calibration linearity was assessed using 10 point calibration curves of 14N tau standard spiked in normal goat serum 0.5%. MRM results showed linearity over 2–32.7 ng/mL concentration range (Table 2). Calibration curves were generated by linear regression analysis by plotting the peak area ratios (14N tau/15N tau) vs. concentration ratios for all measured peptides. The regression coefficients were calculated above 0.98 for the 7 considered peptides. Typical calibration curves are shown in Figure 3. The LOQs were determined over 0.3–2 ng/mL range depending on the peptide (Table 2). Calculated recovery rates were 121 ± 19% for the 7 tau peptides and using the 6.6 ng/mL calibration standard (n = 3). For the TPSLPTPPTR corresponding to the epitope of the ELISA capture antibody, a recovery of 107% was measured.


Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry.

Bros P, Vialaret J, Barthelemy N, Delatour V, Gabelle A, Lehmann S, Hirtz C - Front Neurosci (2015)

Calibration curves of the 7 tau peptides.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555028&req=5

Figure 3: Calibration curves of the 7 tau peptides.
Mentions: Between successive LC-MS/MS analysis, no memory effect phenomenon was observed. The calibration linearity was assessed using 10 point calibration curves of 14N tau standard spiked in normal goat serum 0.5%. MRM results showed linearity over 2–32.7 ng/mL concentration range (Table 2). Calibration curves were generated by linear regression analysis by plotting the peak area ratios (14N tau/15N tau) vs. concentration ratios for all measured peptides. The regression coefficients were calculated above 0.98 for the 7 considered peptides. Typical calibration curves are shown in Figure 3. The LOQs were determined over 0.3–2 ng/mL range depending on the peptide (Table 2). Calculated recovery rates were 121 ± 19% for the 7 tau peptides and using the 6.6 ng/mL calibration standard (n = 3). For the TPSLPTPPTR corresponding to the epitope of the ELISA capture antibody, a recovery of 107% was measured.

Bottom Line: Quantification was performed using isotopically labeled (15)N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs).Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides.This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biochimie et de Protéomique Clinique - Institut de Médecine Régénérative et Biothérapies, Centre Hospitalier Universitaire de Montpellier Montpellier, France ; Laboratoire National de Métrologie et d'Essais (LNE) Paris, France.

ABSTRACT
Tau protein concentration in cerebrospinal fluid (CSF) is currently used as a sensitive and specific biomarker for Alzheimer's disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS) to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid (PCA) followed by micro solid phase extraction (μSPE). We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM) on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labeled (15)N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs). Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

No MeSH data available.


Related in: MedlinePlus