Reprosil pur c18

The LC-MS system consisted of an Easy-nLC 1200 coupled to Q Exactive HF-X mass spectrometer (ThermoFisher scientific, Bremen, Germany) or to Eclipse tribrid mass spectrometer (ThermoFisher Scientific, San Jose, USA). The peptides were eluted on a 450 mm analytical column (8 μm tip, 75 μm ID) packed with ReproSil-Pur C18 (1.9 μm particle size, 120 A pore size, Dr Maisch, GmbH) and separated at a flow rate of 250 nL/min as described in ref. ^{66 (link)}. For DDA measurements, the top 20 most abundant precursor ions selection was performed on the Q Exactive as described^{66 (link)}. For DIA, the Eclipse tribrid mass spectrometer was used to sample ions. The cycle of acquisitions consists of a full MS scan from 300 to 1650 m/z (R = 120,000, ion accumulation time of 60 ms and normalized AGC of 250%) and 22 DIA MS/MS scans in the orbitrap. For each DIA MS/MS scan, a resolution of 30,000, a normalized AGC of 250%, and a stepped normalized collision energy (27, 30, and 32) were used. The maximum ion accumulation was set to auto, the fixed first mass was set to 200 m/z, and the overlap between consecutive MS/MS scans was 1 m/z as described in ref. ^{78 (link)}.

Each sample (equal to 2 µl CSF) was analyzed on a Q-Exactive Plus hybrid mass spectrometer (ThermoFisher Scientific) fitted with a Nanospray Flex ion source and coupled to a NanoAcquity liquid chromatography system (Waters Corporation) essentially as described [20 (link)]. The tryptic peptides were resuspended in 40 μL of loading buffer (2% ACN, 0.1% TFA) and 2 µl was loaded onto a self-packed 1.9 um ReproSil-Pur C18 (Dr. Maisch) analytical column (New Objective, 30 cm × 75 µm inner diameter; 360 µm outer diameter). Elution was performed over a 40-min gradient at a rate of 300 nL/min with buffer B ranging from 2 to 25% (buffer A: 0.1% formic acid in water, buffer B: 0.1% formic acid in ACN). The column was then washed with 99% B for 40 min and re-equilibrated with 2% B for 15 min. The mass spectrometer was set to collect in PRM mode with an inclusion list consisting of each peptide (Additional file 1: Table S3). For PRM scans, the settings were: resolution of 35,000 at 200 m/z, AGC target of 5 × 10⁵ ions, max injection time of 200 ms, loop count of 30, MSX count of 1, isolation width of 1.6 m/z and isolation offset of 0.5 m/z. A pre-optimized normalized collision energy of 28% was used to obtain the maximal recovery of target product ions. A series of product ions from this collision energy optimization were used for downstream peptide quantification.