Collection of CSF samples
The CSF specimens were collected from normal pressure hydrocephalus (NPH) patients, PD patients or cognitively normal healthy control individuals evaluated by investigators at the Johns Hopkins Hospital. The CSF samples from NPH patients were used for method optimization. The CSF samples from PD or control individuals were used to compare pY39 α-syn peptide levels between the two groups. The individuals who are cognitively normal or show PD symptoms were diagnosed after extensive clinical and cognitive testing. All PD patients met the UK Brain Bank criteria for PD diagnosis [11]. After the collection of CSF samples by lumbar puncture, the samples were centrifuged for 10 minutes at 1,500 x g, aliquoted, and stored at -80 °C within one hour of acquisition. The demographic and clinical characteristics of the PD patients and control individuals are shown in Supplemental Table S1.
Sample preparation of cerebrospinal fluids for mass spectrometry analysis
For quantification of pY39 α-syn peptides from PD patients and control samples, 20 fmol of synthetic heavy pY39 α-syn peptide was added to CSF. Cerebrospinal fluid proteins were lysed in 4 M urea and 50 mM triethylammonium bicarbonate (TEAB) followed by a reduction with 10 mM dithiothreitol for 1 h at RT and alkylation with 30 mM iodoacetamide for 30 min at RT in the dark. The proteins were then digested with an endoproteinase Lys-C (1:100; Wako Chemicals, Richmond, VA) by incubating at room temperature for three hours. Sequentially trypsin digestion was conducted by diluting the urea concentration to 2 M by adding 1 volume of 50 mM TEAB followed by adding sequencing-grade trypsin (1:50; Promega, Madison, WI) and incubating at 37o C overnight. The peptide samples were desalted with C18 Sep-Pak (Waters Corporation, Milford, MA) and freeze-dried.
Enrichment of pY39 α-syn peptide
The pY39 α-syn peptides (heavy K8 (13C6/15N2) [EGVLYVGSK*] and endogenous [EGVLYVGSK]) were enriched by performing phosphotyrosine peptide enrichment with PTMScan pY1000 antibody according to manufacturer’s instruction with minor modifications (Cell Signaling Technology, Danvers, MA). Briefly, the CSF peptides derived from 1 ml of CSF were reconstituted in 200 µl of immunoaffinity purification buffer (50 mM MOPS, pH 7.2, 10 mM Na2HPO4 and 50 mM NaCl). The peptide solution was cleared by centrifugation for 5 min at 10,000 x g at 4oC and the supernatant was subject to the phosphotyrosine enrichment. After washing 20 µl of phosphotyrosine agarose beads three times with PBS, the CSF peptide solution was added to the washed beads followed by incubation at 4o C for 2 h with rotation. Subsequently, the supernatant was removed and the beads were washed once with ice-cold water. The bound tyrosine-phosphorylated peptides were eluted by adding 55 µl of 0.15% trifluoroacetic acid (TFA) and incubated at RT for 10 min. After incubation, the tube was centrifuged at 2,000 x g for 1 min and the solution was transferred to a new tube. This elution was repeated once again with 50 µl 0.15% TFA. The eluate was dried using a SpeedVac and the phosphorylated peptides were enriched again using TiO2 beads as described previously [12]. Briefly, 0.6 mg of TiO2 beads (Titansphere) resuspended in 40 µl of binding buffer (65% acetonitrile (ACN) and 2% TFA) were added to the peptides followed by incubation at RT for 20 min with shaking at 1,400 rpm. The peptides were transferred to a C8 StageTip and centrifuged at 2,000 x g for 2 min. Two hundred µl of the washing buffer (65% ACN and 0.1% TFA) was added and centrifuged at 2,000 x g for 5 min. This washing was repeated once again. The phosphopeptides were eluted by adding 40 µl of elution buffer (1% NH4OH and 40% ACN) and centrifuging at 200 x g for 2 min. The eluted peptides were then dried using a SpeedVac followed by reconstitution in 15 µl of 0.1% formic acid prior to mass spectrometry analysis.
Detection of total alpha-synuclein
To normalize the amount of pY39 α-syn peptide in each sample based on the total α-syn present in each sample, the amount of total α-syn in each sample was also measured. Twenty fmol of K8 (13C6/15N2) heavy α-syn peptide (EGVLYVGSK*) for the quantification of the endogenous α-syn peptide were added to 5 µg of CSF peptides followed by desalting with C18 StageTip and LC-MS/MS analysis.
LC-MS/MS analysis
The fractionated peptides were analyzed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer coupled to an EASY-nLC 1200 nano-flow liquid chromatography system (Thermo Fisher Scientific). The peptides from each fraction were reconstituted in 15 µl of 0.1% formic acid and loaded onto an Acclaim PepMap100 Nano-Trap Column (100 µm × 2 cm, Thermo Fisher Scientific) packed with 5 µm C18 particles at a flow rate of 5 µl per min. The flow rate employed was 250 nl/min using a linear gradient of 10–35% solvent B (0.1% formic acid in 95% acetonitrile) over 45 minutes on an EASY-Spray column (50 cm x 75 µm ID, Thermo Fisher Scientific) packed 2 µm C18 particles (Thermo Fisher Scientific), which was fitted with an EASY-Spray ion source operated at a voltage of 2.7 kV. Mass spectrometry analysis was completed in a data-dependent manner with a full scan in the mass-to-charge ratio (m/z) range of 350 to 1,550 followed by targeted MS2. MS1 was measured at a resolution of 120,000 (at m/z of 200). MS2 scan was acquired by fragmenting precursor ions using the higher-energy collisional dissociation (HCD) method and detected at a mass resolution of 30,000 (at m/z of 200). Automatic gain control was set to 500,000 and 100,000 ions for MS1 and MS2, respectively. The maximum ion injection time for MS1 was set to 100 milliseconds. Maximum ion times for MS2 were set to 2,500 and 500 milliseconds for pY39 α-syn and α-syn, respectively. HCD normalized collisional energy (NCE) was set to 25, if not specified. The precursor isolation window was set to 1.6 m/z. Internal calibration was carried out using the lock mass option (m/z 445.1200025) from ambient air. For the light and heavy pY39 α-syn peptides, m/z 516.244 and m/z 520.251 were monitored, respectively. For the light and the heavy Y39 α-syn peptides, m/z 476.261 and m/z 480.268 were monitored, respectively.