Nanoaquity beh130 c18 1.7 μm uplc column

The fraction that contained the major portion of the PgP (Fraction 12) from each of 29 independent gradients (representing material from a total of 87 rats) was pooled. SDS was added to the sample (final concentration = 1%) and the sample concentrated by centrifugation using Amicon 10,000 MWCO concentrators (Millipore, Billerica MA) according to the manufacturer’s protocol. The samples were then mailed to Kendrick Labs (Madison, WI) for further concentration by dialysis, 2-D gel electrophoresis using ampholines in the pH range of 3 –10 and protein identification. The most abundant proteins in the molecular size range of 14–80 kDa were identified by LC-MS/MS using the Kendrick Labs protocol (Darie et al. 2011 (link); Sokolowska et al. 2012a (link); Sokolowska et al. 2012b (link)). Briefly, proteins were identified by digesting the proteins with trypsin and analyzing the peptide mixture by reversed phase liquid chromatography and MS using a NanoAcuity UPLC (Micromass/Waters, Milford, MA) coupled to a Q-TOF Micro MS (Micromass/Waters, Milford, MA). Peptides were loaded onto a 100 μm × 10 nm NanoAquity BEH130 C18 1.7 μm UPLC column (Waters, Milford, MA). Peptides were eluted using a 150 minute gradient of 2–80% organic solvent (acetonitrile containing 0.1% formic acid) with a flow rate of 400 nl/min. The aqueous solvent was 0.1% formic acid in HPLC water.

The extracted peptides mixture was analyzed by reversed phase liquid chromatography (LC) and MS (LC-MS/MS) using a NanoAcuity UPLC (Micromass/Waters, Milford, MA) coupled to a Q-TOF Ultima API MS (Micromass/Waters, Milford, MA), according to published procedures (20 (link)). Briefly, the peptides were loaded onto a 100 μm × 10 mm NanoAquity BEH130 C18 1.7 μm UPLC column (Waters, Milford, MA) and eluted over a 150-minute gradient of 2–80% organic solvent (ACN containing 0.1% FA) at a flow rate of 400 nL/min. The aqueous solvent was 0.1% FA in HPLC water. The column was coupled to a Picotip Emitter Silicatip nano-electrospray needle (New Objective, Woburn, MA). MS data acquisition involved survey MS scans and automatic data dependent analysis (DDA) of the top three ions with the highest intensity ions with the charge of 2+, 3+ or 4+ ions. The MS/MS was triggered when the MS signal intensity exceeded 10 counts/second. In survey MS scans, the three most intense peaks were selected for collision-induced dissociation (CID) and fragmented until the total MS/MS ion counts reached 10,000 or for up to 6 seconds each. Calibration was performed for both precursor and product ions using 1 pmol GluFib (Glu1-Fibrinopeptide B) standard peptide with the sequence EGVNDNEEGFFSAR and the monoisotopic doubly-charged peak with m/z of 785.84.