Proteospin abundant serum protein depletion kit

Two proteins with mass to charge ratios between 12 and 13 kDa were consistently elevated at the 2-week pre-TMA time point by SELDI-TOF, compared to control samples from the equivalent time point. Four additional unique patient pre-TMA serum samples were processed to isolate these proteins. Serum samples were processed using the Proteospin Abundant serum protein depletion kit (Norgen Biotek Corp, ON, Canada) per protocol. The depleted serum was then centrifuged using Millipore 50-kDa centrifugal filter units per kit instructions at 10,000 rpm for 20 min followed by 2 min at 3500 rpm to remove the concentrate. The samples were then subjected to SDS PAGE using Novex 18% tris-glycine1.0 mm gels, and Coomassie Blue staining. Proteins in the 12–17-kDa range that were overexpressed in the pre-TMA samples compared to baseline samples were excised from the gel. The gel sections were reduced, alkylated, and digested with trypsin. The resulting peptides were recovered and analyzed by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the Linear Trap Quadropole (LTQ). A MASCOT search of homo-sapiens was used to identify the proteins. Two haptoglobin fragments were identified among the proteins isolated in the 12–17-kDa gel sections from pre-TMA sera.