The goal of this study was to evaluate the sensitivity, reproducibility and robustness of the PepFinder Kit, using the Finnigan LCQ Deca XP Plus ion trap mass spectrometer. The ability of the Finnigan Surveyor HPLC system, used with an optimized splitting system, to deliver consistent flow at flowrates rates of 100200 nL/min was also evaluated.
Protein standards human serum albumin, human IgG, human transferrin, human hemoglobin, human macroglobulin, and horse myoglobin were purchased from Sigma-Aldrich, St. Louis, MO, USA.
Reduction, alkylation and digestion
One mg of each lyophilized protein standard was reconstituted separately in 1 mL of ammonium bicarbonate buffer (100 mM, pH 8.5) and 3 μL DTT (1 M, Sigma- Aldrich, St. Louis, MO, USA). The mixture was incubated for 30 minutes at 37C. To alkylate the protein, 7 μL of iodoacetic acid (1 M in 1 M KOH, Sigma-Aldrich, St. Louis, MO, USA) was added and the mixture was incubated for an additional 30 minutes at room temperature in the dark. Thirteen μL DTT (1 M) was added to quench the iodoacetic acid. The reduced and alkylated proteins were digested by adding 20 μL modified trypsin (0.5 mg/mL, Promega, Madison, WI, USA). The mixture was incubated for 6 hours at 37C, then an additional 20 μL trypsin (0.5 mg/mL) was added and incubation was continued for 16 hours at 37C.
The digested samples were analyzed using a fully automated nanoflow LC/MS/MS system, configured with a PepFinder Kit (Figure 1). Aliquots of 10 μL were placed in wells of a 96-well plate (Nalg