The brevican/neurocan panel has previously been described in detail [30 ]. Briefly, in total 20 isotope-labelled tryptic peptides (nine for brevican and eleven for neurocan), labelled with 13C and 15 N at the C-terminal arginine or lysine, were used as internal standard (IS) peptides (JPT Peptide Technologies, Berlin, Germany) (Additional file 1 : Table S1). Twenty-five μL of the IS mixture was spiked into 100 μL CSF, followed by reduction, alkylation, trypsin digestion and subsequent sample clean-up.
Next, the dried samples were reconstituted in 100 µL 50 mM ammonium bicarbonate. Samples were then analysed by high-performance liquid chromatography (HPLC)—mass spectrometry (MS)-based method with a parallel reaction monitoring (PRM) approach, using a Vanquish UHPLC coupled to a high resolution Q Exactive hybrid quadrupole-orbitrap mass spectrometer, with electrospray ionization (Thermo Fisher Scientific), operated as previously described [30 , 31 (link)]. Each sample (50 µL) was loaded onto a Hypersil Gold reversed phase HPLC C18 column (Thermo Fisher Scientific) operated at a flow rate of 300 µL/min. Mobile phases were A: 0.1% formic acid in deionized water (v/v) and B: 0.1% formic acid/84% acetonitrile in deionized water (v/v/v). The applied gradient was 0 to 40% B over 20 min.
Next, the dried samples were reconstituted in 100 µL 50 mM ammonium bicarbonate. Samples were then analysed by high-performance liquid chromatography (HPLC)—mass spectrometry (MS)-based method with a parallel reaction monitoring (PRM) approach, using a Vanquish UHPLC coupled to a high resolution Q Exactive hybrid quadrupole-orbitrap mass spectrometer, with electrospray ionization (Thermo Fisher Scientific), operated as previously described [30 , 31 (link)]. Each sample (50 µL) was loaded onto a Hypersil Gold reversed phase HPLC C18 column (Thermo Fisher Scientific) operated at a flow rate of 300 µL/min. Mobile phases were A: 0.1% formic acid in deionized water (v/v) and B: 0.1% formic acid/84% acetonitrile in deionized water (v/v/v). The applied gradient was 0 to 40% B over 20 min.