Nano advance

Total protein concentration in CSF was determined by using the 2D Quant kit (GE Healthcare Life Sciences, UK), according to the manufacturer’s protocol, and 400 µg total protein was used as input for profiling. All samples were loaded on an affinity removal column for the depletion of the 14 most abundant proteins (MARS-14, Agilent Technologies, Santa Clara, CA, USA). After tryptic digestion, CSF samples were fractionated in 20 fractions using high pH reversed-phase C18 LC and each fraction was subsequently analyzed by nanoflow liquid chromatography (Bruker Daltonics; nano-Advance) connected online to an ultra-high resolution quadrupole time-of-flight tandem mass spectrometer (Qq-TOF; Bruker Daltonics; maXis 4G ETD) as described previously^{50 (link)}.
Raw MS data were analyzed by MaxQuant software version 1.5^{51 (link)} with pre-defined Qq-ToF parameter settings against the RefSeq (release 55) human protein sequence database. We set cysteine carbamidomethylation as a fixed modification, whereas N-terminal acetylation, methionine oxidation, and deamidation of glutamine and/or asparagine were set as variable modifications. For further statistical analysis, only peptides with intensity above the detection limit in at least 75% of the samples in one of the groups (PD, MSA, or non-neurological controls) were used.

The washed protein pellet was dissolved in 100 μL 50 mM ammonium bicarbonate by sonication for 5 minutes. The dissolved proteins were then reduced with 50 mM tris-(2-carboxyethyl) phosphine (TCEP), alkylated with 360 mM acrylamide and finally digested with sequencing grade trypsin (Promega). Nanoflow LC-MS/MS was performed on a Nano-Advance (Bruker) HPLC. Samples were loaded onto a C₁₈ trap column and then switched in-line with an analytical column (Bruker, 0.1 x 150 mm Magic C₁₈ AQ 3.0μm, 200Å). Elution was performed at 0.8 μL/min, using a tailored gradient from 0%-35% acetonitrile (with 0.1% formic acid) over 60 minutes and then from 35%-45% acetonitrile (with 0.1% formic acid) in 10 minutes. The column outlet was directly interfaced to an amaZon Speed ETD (Bruker) mass spectrometer. Automated information dependent acquisition (IDA) was performed using Hystar PP 3.2.44.0 software, with a MS survey scan over the range m/z 350–1200 followed by three MS/MS spectra from 50–3000 m/z acquired during each cycle of 30 ms duration.

The lyophilized peptide fractions were reconstituted in 0.1% formic acid in LC-MS-grade water and subjected to nano-LC (Nano-Advance; Bruker, Germany) followed by identification in captive ion source (Bruker Captive Spray tip) spray-in Maxis-HD qTOF (Bruker) mass spectrometer (MS) with high mass accuracy and sensitivity. The peptides were enriched in nano-trap column (Acclaim Pep Map, particle size 5 μm, pore size 100 Å; Thermo Scientific) and eluted on to nano-analytical column (Kaya Tech HIQ SIL C₁₈HS/3, 0.1 × 150 mm, 3 μm particle size, and 200 Å pore size). The peptide elution was carried out using a linear gradient of 5–45% acetonitrile at 400 nl/min flow rate in a total run time of 135 keeping the solvent system as follows: solvent A, 100% water in 0.1% formic acid; and solvent B, 100% acetonitrile in 0.1% formic acid. Positive ions were generated by electrospray, and the q-TOF was operated in data-dependent acquisition mode to automatically switch between MS and MS/MS acquisition. Precursor ion TOF MS survey scan was acquired with a range of 300–1,800 m/z with resolution R = 75,000. Q1 sequentially selects six most intense precursor ions for fragmentation using collision-induced dissociation for MS-MS analysis with a fixed cycle time of 3 s along with 2 min of release for exclusion filter (Data acquisition otof software, version 24.8; Bruker Daltonics).

Nanoflow electrospray ionization tandem mass spectrometry (Nano ESI-MS) analysis was carried out using a Bruker nanoLC system (Nano Advance, BrukerDaltonics) along with a captive spray- Maxis-HD-qTOF mass spectrometer (BrukerDaltonics) with high accuracy and sensitivity. Digested peptides were desalted, concentrated on C18 reverse phase ZipTips, and eluted twice with 20 μL 60% acetonitrile in 0.1% formic acid before vacuum drying at room temperature. Peptides were resuspended in 10 μL 0.1% formic acid, and subjected to nLC-MS/MS. Peptides were separated using 15 cm long analytical columns (Bruker Magic C18AQ, 0.1 × 150 mm, 3 μm particle size and 200 Å pore size) of 135 minute gradient from 5% to 90% acetonitrile in 0.1% formic acid and a flow rate of 400 nL per minute. The mass spectrometer was operated in data-dependent acquisition mode with 3 intense precursor ions. Exclusion of all charges less than 2 and greater than 6 were used, and all unknown charges were kept in rejection mode. A dynamic exclusion of 20 seconds was used. Full mass spectrometry spectra scans were acquired with a scan range from 350 to 2200 m/z.

Peptide digests were analyzed by electrospray ionization in the positive mode on an ion trap instrument Amazon Speed (Bruker, Bremen, Germany), using captive spray source. Two analytical replicates of every sample were done. Peptides were separated by nanoflow HPLC (NanoAdvance, Bruker, Bremen, Germany). UHPLC Nanotrap (100 μm i.d. × 25 mm long) packed with 200 A C₁₈ stationary phase (5 μm, C18AQ, Michrom) was used for peptide trapping. Analytical columns (100 μm × 150 mm long) packed with 200 A C₁₈ stationary phase (3 μm, C18AQ, Michrom) were coupled to the mass spectrometer (MS).
Peptide mixtures obtained after tryptic digestion were applied to the precolumn at a flow rate of 5 μL min⁻¹ in 2% (v/v) acetonitrile with 0.1% (v/v) formic acid. Peptides were eluted by a linear gradient of A (water, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid), as follows: 0 min – A (98%), B (2%); 50 min – A (5%), B (95%); 50–55 min – A (5%), B (95%) with flow rate of 400 nL min⁻¹. Ion source conditions were optimized with a calibration solution according to the instrument provider. All MS survey scans were performed from m/z 400–1400 with enhanced resolution. Data analysis was performed by selection of the five most abundant precursors rejecting singly charged ions.