Pepmap100 c18 column

For library preparation, pooled protein extracts from a mix of FTD-MAPT and NDC samples were used. Extracted peptides were analysed by micro liquid chromatography with tandem mass spectrometry (LC–MS/MS) using an Ultimate 3000 LC system (Dionex, Thermo Scientific) coupled to the TripleTOF 5600 mass spectrometer (Sciex). Peptides were trapped on a 5 mm Pepmap 100 C18 column (300 μm i.d., 5 μm particle size, Dionex) and fractionated on a 200 mm Alltima C18 column (300 μm i.d., 3 μm particle size). The acetonitrile concentration in the mobile phase was increased from 5 to 18% in 88 min, to 25% at 98 min, 40% at 108 min and to 90% in 2 min, at a flow rate of 5 μL/min. The eluted peptides were electro-sprayed into the TripleTOF MS with a micro-spray needle voltage of 5,500 V. The mass spectrometer was operated in a data-dependent acquisition (DDA) mode with a single MS full scan (m/z 350–1250, 150 ms) followed by a top 25 MS/MS (m/z 200–1800, 150 ms) at high sensitivity mode in UNIT resolution, precursor ion > 150 counts/s, charge state from + 2 to + 5, with an exclusion time of 16 s once the peptide was fragmented. Ions were fragmented in the collision cell using rolling collision energy, and a spread energy of 5 eV.

Peptides of the individual sample fractions were dissolved in 12 µL of 0.1% (v/v) acetic acid. In the first set, the sample was loaded onto an Ultimate 3000 LC system (Dionex, Thermo Scientific). Peptides were trapped on a 5 mm Pepmap100 C18 column (Dionex) and fractionated on a 200 mm Alltima C18 column (100 μm id, 3 μm particle size). In the second set, the sample was loaded onto a nanoLC 425 system (Sciex) and fractionated on a 120 mm C18 column (150 µm id column packed with 1.9 µm Reprosil-Pur 120 C18-AQ beads). Acetonitrile concentration in the mobile phase in 0.1% formic acid was increased from 5 to 18% in 88 min, to 25% at 98 min, 40% at 108 min, and to 90% at 110 min. The flow rate was 400nL/min. Peptides were electrosprayed into an SCIEX TripleTOF® 5600 mass spectrometer using an ion spray voltage of 2.5 kV, curtain gas at 35 p.s.i., nebulizer gas at 15 p.s.i., and an interface heater temperature of 150 °C. The MS survey scan range was m/z 350–1250 acquired for 250 ms. The top 20 precursor ions were selected for 85 ms per MS/MS acquisition, with a threshold of 90 counts. Dynamic exclusion was 16 s. Rolling CID function was activated, with an energy spread of 15 eV. Analysis of one sample (tangle bearing neurons) failed due to technical problems and was removed from the analysis leaving an n = 11 for the tangle bearing neurons.

Peptides were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 LC system (Dionex, Thermo Scientific) coupled to the Triple TOF 5600 mass spectrometer (Sciex). Peptides were trapped on a 5-mm Pepmap 100 C18 column (300 μm i.d., 5 μm particle size, Dionex) and fractionated on a 200-mm Alltima C18 column (300 μm i.d., 3 μm particle size). The acetonitrile concentration in the mobile phase was increased from 5 to 18% in 88 min, 25% at 98 min, 40% at 108 min, and 90% in 2 min, at a flow rate of 5 μL/min. The eluted peptides were electro-sprayed into the Triple TOF MS. The micro-spray needle voltage was set to 5500 V. SWATH experiments consisted of a parent ion scan of 150 ms followed by a SWATH window of 8 Da with a scan time of 80 ms and stepped through the mass range between 450 and 770 m/z. The total cycle time was about 3.2 s, which yielded in general 9–10 measurement points across a typical peptide with an elution time of 30 s. The collision energy for each window was determined based on the appropriate collision energy for a 2+ ion, centred upon the window with a spread of 15 eV.

Peptides were quantified by LC–MS/MS using an Ultimate 3000 LC system (Dionex, Thermo Scientific) coupled to a Triple TOF 5600 mass spectrometer (Sciex). Peptides were trapped on a 5 mm Pepmap 100 C18 column (300 µm i.d., 5 µm particle size, Dionex) and fractionated on a 200 mm Altima C18 column (100 µm i.d., 3 µm particle size). The concentration of acetonitrile in the mobile phase was increased from 5–18% in 88 min, 18–25% in 98 min, 25–40% in 108 min and to 90% in 2 min at a flow rate of 5 µL/min. Eluted peptides were electro-sprayed into the TripleTOF MS using a microspray needle of 5500 V. SWATH experiments consisted of a parent ion scan of 150 ms followed by a SWATH window of 8 Da with a scan time of 80 ms. It was stepped through the mass range between 450 and 770 m/z. The collision energy per window was based on the appropriate collision energy for 2 + ions centered upon the window with a spread of 15 eV.

Nano-LC-MS/MS analysis on tryptic-digested UTI bands was performed for protein identification and characterization as previously described [31 , 32 (link)]. Briefly, spots were excised, destained, and dehydrated. Rehydrated spots were in gel reduced, alkylated, and trypsin digested. Recovered peptides were purified through a C18 column, and 2 μL was injected into a nano-HPLC system (Eksigent, ABSciex, USA). Peptides were separated through a C18 PepMap-100 column (3 μm, 75 μm × 250 mm, Thermo Scientific, USA) in a 70-minute linear gradient from 5% of 0.1% formic acid to 40% of acetonitrile/0.1% formic acid. HPLC was directly coupled to a TripleTOF™ 5600 mass spectrometer (ABSciex, USA), and MS/MS data were processed with ProteinPilot™ Software (ABSciex, USA).