Quantitative proteomics of the chemokine IL-8 applying orbitrap and triple quadrupole mass spectrometer

Proteome profiling in the background of a biological matrix requires sensitive, accurate and reproducible measurement of a large number of proteins in a single run. In case of low abundant proteins such as chemokines this is especially challenging due to the high dynamic range of protein concentrations and the high complexity in the sample. In this study we assessed the analytic performance of two currently most used technics on the proteomics sector for quantification: SRM as targeted approach, using a triple quadrupole instrument; and shotgun proteomics as high throughput protein identification approach, using a quadrupole-orbitrap instrument. The low abundant chemokine Il-8 was used as a target protein, which had to be identified and quantified in complex biological matrices. Matrix effects were investigated by spiking IL-8 in three different matrices: 1) phosphate buffered saline (PBS); 2) human serum and 3) supernatant of human umbilical vein endothelial cells (HUVECs) grown in endothelial basal medium (EBM-2) supplemented with 10% fetal calf serum (FCS). Furthermore, we investigated the benefit of protein enrichment, using immunoprecipitation of IL-8 from all three matrices. As a conclusion, immunoprecipitation as a tool for sensitive protein enrichment and SRM as targeted proteomics method aimed for highly sensitive and selective detection seem to give a promising combination for a sensitive, accurate and reliable quantitative determination of low abundant proteins in complex matrices.