Proteins from concentrated Pap test and swab samples were trypsin digested and prepared for MS by the Filter Aided Sample Preparation (FASP) method using Nanosep Omega centrifugal devices with a 10K MW cut off (Pall Corp., Port Washington, NY) as previously described [8 (link)]. Concentrated Pap test and swab samples (~ 50 µg protein) were solubilized in 10 mM Tris, pH 7.6, 0.4% SDS, and reduced by the addition of 10 mM TCEP at room temperature, alkylated with 50 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO) and digested overnight at 37 °C with sequencing grade trypsin (Promega, Madison, WI) using an enzyme:protein ratio of 1:100. Peptides were desalted with C18 stage tips (Thermo Scientific, West Palm Beach, FL) and dried under vacuum.
Tumor tissue proteins were digested “in solution” as follows: 200 µg of the tumor tissue extract was diluted five-fold with ultra-pure water. Trypsin was added in a 1:40 ratio of trypsin to total protein. The sample was incubated for 16 h at 37 °C. After incubation, the sample was frozen at − 80 °C for 30 min and dried in a vacuum centrifuge. The sample was then cleaned with a 4 ml Extract Clean™ C18 SPE cartridge from Grace–Davidson (Deerfield, IL) and the eluate was vacuum dried.
Tumor tissue proteins were digested “in solution” as follows: 200 µg of the tumor tissue extract was diluted five-fold with ultra-pure water. Trypsin was added in a 1:40 ratio of trypsin to total protein. The sample was incubated for 16 h at 37 °C. After incubation, the sample was frozen at − 80 °C for 30 min and dried in a vacuum centrifuge. The sample was then cleaned with a 4 ml Extract Clean™ C18 SPE cartridge from Grace–Davidson (Deerfield, IL) and the eluate was vacuum dried.