6490 triple quadrupole mass spectrometer

The concentrations of PA, TYR, HPPA, HPLA and HGA in serum and urine were measured by liquid chromatography tandem mass spectrometry^{16 (link),18 (link)}. The published method was further validated to include PA, HPPA and HPLA (unpublished data). All analyses were performed on an Agilent 6490 Triple Quadrupole mass spectrometer with Jet-Stream electrospray ionisation coupled with an Agilent 1290 Infinity II Ultra High Performance Liquid Chromatography pump and autosampler. Briefly this method incorporates reverse-phase chromatographic separation on an Atlantis dC18 column (100 mm × 3.0mm, 3 µm, Waters); initial chromatographic conditions of 80:20 water:methanol with 0.1% formic acid (v/v) increased linearly to 10:90 over 5 min. Matrix-matched calibration standards and quality controls were used with appropriate isotopically-labelled internal standards with quantitation in multiple reaction mode (PA and TYR in positive ionisation and HPPA, HPLA and HGA in negative ionisation). Sample preparation was by dilution in a combined internal standard solution containing ¹³C₆-HGA, d₄-TYR and d₅-PA in 0.1% formic acid (v/v) in deionised water. Concentrations were adjusted to reflect the concentration differences between serum and urine samples. No internal standard was available for HPPA and HPLA at time of analysis and so ¹³C₆-HGA was validated for use as the internal standard.

Targeted proteomics was performed according to our recently published method [12 (link)]. Prior to LC-MS/MS analysis, four synthetic standard peptides were added to each sample (10 fmol/μL each) including Glu1-Fribrinopeptide B (Peptide Sequence: EGVNDNEEGFFSAR), M28 (Peptide Sequence: TTPAVLDSDGSYFLYSK), HK0 (Peptide Sequence: VLETKSLYVR) and HK1 (Peptide Sequence: VLETK(ε-AC)SLYVR). The trypsin digested supernatants were analyzed by a nano-Chip-LC using a 1260 Infinity Series HPLC system (Agilent) coupled to a 6490 triple quadrupole mass spectrometer (Agilent). A large capacity protein chip (G4240-62010) with a 160 nL enrichment column and a 150 mm x 75 μm separation column (5 μm ZORBAX 300SB-C18, 30 Å pore size) was used and 1 μL of the sample was injected. Mobile phase A consisted of 97.8% H₂O, 2% ACN and 0.2% FA, mobile phase B of 99.8% acetonitrile and 0.2% formic acid. A flow rate of 5 μL/min was applied for sample loading via the capillary pump and 400 nL/min for peptide separation via the nano pump. A 20 min gradient was applied for peptide separation while the total run time was 40 min including column regeneration. Total peak areas of peptides were exported from Skyline (Version 2.5, Ref. [17 (link)]) as csv-files and normalization was performed in Excel.