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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

Sample extraction: 15N recombinant tau was spiked on CSF and proteins were then precipitated. Remaining proteins including tau in the supernatant were extracted on HLB cartridges. During extraction, an oxidation step was performed to oxidize methionine. Extracts were dried and then enzymatically digested by trypsin. Proteotypic peptides of tau protein were then quantified in the sample by LC-MS/MS.
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Figure 1: Sample extraction: 15N recombinant tau was spiked on CSF and proteins were then precipitated. Remaining proteins including tau in the supernatant were extracted on HLB cartridges. During extraction, an oxidation step was performed to oxidize methionine. Extracts were dried and then enzymatically digested by trypsin. Proteotypic peptides of tau protein were then quantified in the sample by LC-MS/MS.

Mentions: Sample extraction of tau peptides were performed according to Barthelemy et al. (Barthelemy, under review). Briefly, 450 μL of CSF or 0.5% serum samples were mixed with 50 μL of 15N-tau-441 (50 ng/mL) in a LoBind tube. Protein precipitation was performed by adding 25 μL of 70% PCA. Samples were then vortexed, kept on ice for 15 min before centrifugation at 17,000 g at 4°C during 15 min to obtain a clear supernatant. Supernatants were acidified with 50 μL of 1% trifluoroacetic acid (TFA). SPE with a hydrophilic-lipophilic balance SPE 96-well plate was conditioned with 300 μL of MeOH and equilibrated with 500 μL of 0.1% TFA. Samples were loaded and washed with 500 μL of 0.1% TFA. For protein oxidation, 500 μL of 3% FA and 3% H2O2 solution in water was loaded on cartridge and kept 12 h at 4°C. Thereafter, cartridge was washed with 500 μL of 0.1%TFA. Oxidized tau proteins were eluted with 100 μL of 35% acetonitrile 0.1% TFA. Extracts were evaporated to dryness with a Speedvac instrument from LabConco (Kansas City, MO, USA) and resuspended with 40 μL of 1 ng/μL trypsin solution in 50 mM ammonium bicarbonate. The digestion was performed for 24 h at 37°C on a Thermomixer R from Eppendorf (Hambourg, Germany) and stopped with 5 μL of 10% FA and stored at −20°C prior to LC-MS/MS analysis (Figure 1).


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)

Sample extraction: 15N recombinant tau was spiked on CSF and proteins were then precipitated. Remaining proteins including tau in the supernatant were extracted on HLB cartridges. During extraction, an oxidation step was performed to oxidize methionine. Extracts were dried and then enzymatically digested by trypsin. Proteotypic peptides of tau protein were then quantified in the sample by LC-MS/MS.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Sample extraction: 15N recombinant tau was spiked on CSF and proteins were then precipitated. Remaining proteins including tau in the supernatant were extracted on HLB cartridges. During extraction, an oxidation step was performed to oxidize methionine. Extracts were dried and then enzymatically digested by trypsin. Proteotypic peptides of tau protein were then quantified in the sample by LC-MS/MS.
Mentions: Sample extraction of tau peptides were performed according to Barthelemy et al. (Barthelemy, under review). Briefly, 450 μL of CSF or 0.5% serum samples were mixed with 50 μL of 15N-tau-441 (50 ng/mL) in a LoBind tube. Protein precipitation was performed by adding 25 μL of 70% PCA. Samples were then vortexed, kept on ice for 15 min before centrifugation at 17,000 g at 4°C during 15 min to obtain a clear supernatant. Supernatants were acidified with 50 μL of 1% trifluoroacetic acid (TFA). SPE with a hydrophilic-lipophilic balance SPE 96-well plate was conditioned with 300 μL of MeOH and equilibrated with 500 μL of 0.1% TFA. Samples were loaded and washed with 500 μL of 0.1% TFA. For protein oxidation, 500 μL of 3% FA and 3% H2O2 solution in water was loaded on cartridge and kept 12 h at 4°C. Thereafter, cartridge was washed with 500 μL of 0.1%TFA. Oxidized tau proteins were eluted with 100 μL of 35% acetonitrile 0.1% TFA. Extracts were evaporated to dryness with a Speedvac instrument from LabConco (Kansas City, MO, USA) and resuspended with 40 μL of 1 ng/μL trypsin solution in 50 mM ammonium bicarbonate. The digestion was performed for 24 h at 37°C on a Thermomixer R from Eppendorf (Hambourg, Germany) and stopped with 5 μL of 10% FA and stored at −20°C prior to LC-MS/MS analysis (Figure 1).

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