Tryptic peptides from pixelation ofthe fractionated plasma sample were analyzed on an LCQ DecaXPI ITmass spectrometer (Thermo Electron, San Jose, CA, USA) interfaced with a MicroPro pump (Eldex, Napa, CA, USA) and an autosampler. Serum tryptic digests were analyzed on an LTQ linear IT mass spectrometer (Thermo Electron) coupled with a NanoLC pump (Eksigent Technologies, Liver-more, CA, USA) and autosampler. For each pixel, 5 mL (plasma samples) or 7 mL (serum samples) ofthe tryptic digest (total ~30 mL) was analyzed. Tryptic peptides were separated by RP-HPLC on a nanocapillary column, 75 mm id x 20 cm Pico-Frit (New Objective, Woburn, MA, USA), packed with MAGIC C18 resin, 5 mm particle size (Michrom BioResources, Auburn, CA, USA). In some ofthe initial optimization experiments, POROS R2 C18 resin, 10 mm particle size (Applied Biosystems, Foster City, CA, USA) was used. Solvent A was 0.58% acetic acid in Milli-Q water, and solvent B was 0.58% acetic acid in ACN. Peptides were eluted into the mass spectrometer at 200 nL/min using an ACN gradient. Each RP-LC run consisted of a 10 min sample load at 1% B; a 75 min total gradient consisting of 128% B over 50 min, 28-50% B over 14 min, 50-80% B over 5 min, 80% B for 5 min before returning to 1% B in 1 min. To minimize carryover, a 36 min blank cycle was run between each sample. Hence, the total sample-to-sample cycle time was 121 min. In some optimization experiments, a 49 min gradient (1-28% B over 27 min, 28-50% B over 11 min, 50-80% B over 5 min, 80% B for 5 min before returning to 1% B in 1 min) was used instead ofthe 75 min gradient.
The mass spectrometers were set to repetitively scan m/z from 375 to 1600 followed by data-dependent MS/MS scans on the three most intense (LCQ Deca XP +) or the ten most abundant (LTQ) ions with dynamic exclusion enabled. In some experiments, gas-phase fractionation using different m/z ranges was performed as described in Fig. 2B.
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