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 previously50 (link).
Raw MS data were analyzed by MaxQuant software version 1.551 (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.
Raw MS data were analyzed by MaxQuant software version 1.551 (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.
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