We tested both nanoflow and standard flow LC mass spectrometry method for the application of MRM-based protein quantitiation to large epidemiological cohorts. By using the standard flow LC method, we have reduced the LC-MS run time to almost a third of the nanoflow LC-MS approach. Based on a comparison of the quantification of 100 plasma proteins in more than 1500 LC-MS runs, the standard deviation range of the retention time during continuous operation was substantially lower with the standard flow LC-MS (<0.05 min) compared to the nanoflow LC-MS method (0.26-0.44 min). In addition, the standard flow LC method also offered less variation in protein measurements. However, five times more sample volume was required to achieve similar sensitivity.

Plasma samples were analyzed using the PlasmaDive MRM Panel (Biognosys AG, CH). Sample processing was semi-automated on a Bravo liquid handling system (Agilent Technologies, US). Briefly, 10μl of plasma samples were denatured, reduced and alkylated according to the manufacturer’s instruction, followed by spiking-in of SIS peptides and an in-solution digestion overnight using trypsin (Pierce). After solid phase extraction clean-up with a 96-well C18 spin plate (Harvard apparatus), the eluted peptides were dried using SpeedVac (Thermo Fisher Scientific) and resuspended in 40μl of LC solution with addition of indexed retention time peptides to adjust the retention time variability across different LC-MS runs. A pooled sample was used as QC sample with repeated injections during continuous operation. On the nanoflow platform (Thermo U3000 RSLCnano, Thermo Fisher Scientific), 2μl of digested samples were directly injected onto a 15cm column (AcclaimPepMap100, C18, 15cm x 75um, 3um, 100Å) and separated over a 32min gradient at 300nl/min (0-30min, 5%-35% B; 30-32min, 35%-99% B; 32-40min, 99% B; 40-70min, 2% B; A=1% acetonitrile, 0.1% formic acid; B=97% acetonitrile, 0.1% formic acid). The nanoflow LC was interfaced to a TSQ Vantage mass spectrometer (Thermo Fisher Scientific). Analysis was performed using the following parameters: Q1 resolution 0.7Th, Q2 collision gas pressure 1mTorr, Q3 resolution 0.7Th. 850 transitions were exported from SpectroDive software version 6 (Biognosys AG, CH) and scheduled with a cycle time of 3 seconds and a retention time (RT) window of 4min. All the SIS peptides were labelled with heavy Lysine (+8) or Arginine (+10) and the cysteines were carbamidomethylated (+57). The same samples were also analysed on a standard flow platform (Agilent 1290 Infinity II LC). 10μl of digests were directly injected onto a 25cm column (AdvanceBio Peptide Mapping, C18, 2.1mm x 250mm, 2.7um, 120Å) and separated over a 23min gradient at 350μl/min (0-0.5min, 5%-7.5% B; 0.5-18min, 7.5%-28% B; 18-20min, 28%-95% B; 20-23min, 95% B; 23-27min, 5% B; A=0.1% formic acid in H2O; B=0.1% formic acid in acetonitrile) at 50C. The standard flow LC was interfaced to an Agilent 6495 QqQ mass spectrometer and both were controlled by MassHunter Workstation software (version B.08.00). Both Q1 and Q3 were set at Unit resolution (0.7Th) and the following parameters were used: Delta EMV 350, Frag 380V, Cell Acc 4V, Gas Temp 200C, Gas Flow 11L/min, Nebulizer 35psi, Sheath Gas Heater 250C, Sheath Gas Flow 12L/min, Capillary 4kV, VCharging 300, Ion Funnel Pos High Pressure RF 180V and Pos Low Pressure RF 90V. 765 transitions were scheduled using Dynamic MRM with a cycle time of 0.5 second and a RT window of 0.8 min.

A subset (n=44) of Bruneck study year 2000 samples.