Selective Fishing for Peptides with Antibody-Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS

An online microfluidics-mass spectrometry platform was developed for determining proteotypic peptides from in-solution digested samples. Accelerated and selective sample cleanup was achieved by integrating proteotypic epitope peptide immunoextraction with nano liquid chromatography-tandem mass spectrometry (online IE-nanoLC-MS/MS). Ten individually prepared 180 mu m inner diameter capillaries with ethylene glycol dimethacrylate-co-vinyl azlactone (EDMA-co-VDM) monoliths were immobilized with anti-protein antibodies that are used in routine immunoassays of the intact small cell lung cancer biomarker ProGRP. The resulting AB columns provided linearity correlation coefficients of 0.960.99 for protein amounts and concentrations of 10 pg to 5 ng and 0.5-250 ng/mL, respectively. The columns/platform gave relative peak area RSDs below 15%. The IE-nanoLC-MS/MS platform provided a limit of detection (LOD) of 520 pg/mL of ProGRP in human serum. The approach was applicable for other matrixes and proteins, i.e., primary glioblastoma cells and endogenous alpha V integrin chain. Thus, EDMA-co-VDM monoliths immobilized with antibodies are suited for automated peptide capture in microfluidic formats.