Nanoacquity ultraperformance lc system

Retention times of the 61 peptides were checked, and S/N for the 122 transitions were assessed by synthesizing isotopically labelled SpikeTides comprising C‐terminal heavy Lys (¹³C₆¹⁵N₂‐Lys) or Arg (¹³C₆¹⁵N₄‐Arg; JPT Peptide Technologies GmbH). JPT also commercialized a pool of the isotope‐labelled peptides to support the rapid setup of targeted assays. A total of 0.1 pmol of each SpikeTide_L was added to 10 μg of an Arabidopsis digested total protein extract and analysed using a 25‐min gradient at 500 μl min⁻¹ with a 6600 TripleTOF (SCIEX) and a nanoACQUITY UltraPerformance LC system (Waters), incorporating a C18 reverse phase column (SCIEX, 0.3 × 150 mm, 3 μm particle size). Buffer composition and gradient formation was as for SRM assays above.

All samples from an individual were run in the same batch. CSF tau was immunopurified and digested, then phosphorylated and nonphosphorylated peptides were quantified using high-resolution MS (HRMS) as previously described and detailed in Appendix 1 (ref. ²⁵ ). Briefly, tau was immunopurified by incubating 450 µl of CSF with tau1 and HJ8.5 antibodies covalently attached to Sepharose beads at room temperature for 4 h. Immunopurified tau was digested for 16 h at 37 °C with 400 ng of trypsin (Promega). AQUA peptides (Life Technologies) were added to a final sample concentration of 5 fmol per labeled phosphorylated peptide and 50 fmol per labeled unmodified peptide. Samples were subjected to liquid chromatography–tandem HRMS (LC-MS/HRMS) analysis on a nanoAcquity ultra-performance LC system (Waters) coupled to an Orbitrap Tribrid Eclipse mass spectrometer (Thermo Fisher Scientific) operating in parallel reaction monitoring mode. MS/HRMS transitions were extracted using Skyline v.22.2.2.278 (MacCoss lab, University of Washington). Data were aggregated using Tableau v.2022.2.2 (Tableau Software) to calculate concentrations and phosphorylation occupancies. All assays and data extraction steps were performed by operators blinded to any clinical or biomarker information.

Protein identification was conducted in the same way as described in [8 ]. Measurements were performed using a nanoAcquity UltraPerformance LC system connected to an auto-sampler equipped with a HSS T3 analytical column (1.8-µm particle, 75 µm × 150 mm) kept at 45 °C, and a Symmetry C18 trap column (5-µm particle, 180 µm × 20 mm), all Waters, USA. This setup was connected to an LTQ Orbitrap Elite. A 180-min gradient was used: (0–5 min: 99% buffer A and 1% buffer B, 5–10 min 99–94% A, 10–161 min: 94–60% A, 161–161.5 min: 60–14% A, 161.5–166.5 min: 14–4% A, 166.5–167.1 min: 99% A, 167.1–180 min: 99% A). To optimize the method for small cell numbers, the MS/MS max ion inject time was increased to 400 ms.