Reducing Protein Oxidation in Low-Flow Electrospray Enables Deeper Investigation of Proteoforms by Top Down Proteomics

Publication date: Available online 5 June 2015 Source:EuPA Open Proteomics Author(s): Kyunggon Kim , Philip D. Compton , Timothy K. Toby , Paul M. Thomas , John T. Wilkins , R.Kannan Mutharasan , Neil L. Kelleher Enabling the implementation of top down proteomic techniques within clinical workflows requires a dramatic increase in sensitivity. It has been previously demonstrated that electrospray ionization (ESI) becomes more efficient with decreasing volumetric flow rates at the emitter. Therefore, narrow inner diameter (I.D.) columns used in front-end chromatographic separations yield increased sensitivity. However, the smaller cross-sectional area of a narrow I.D. column places a larger fraction of the eluent in fluid communication with the electrode within the high voltage union that facilitates electrospray ionization (ESI), leading to increased oxidation of solution-phase proteins. Oxidation of proteins alters their chemical state of the protein, complicates data analysis, and reduces the depth of proteome coverage attained in a typical top-down proteomics experiment. Excessive protein oxidation results in poor deconvolution and exact mass calculations from MS1 spectra, interferes with peak isolation for MS/MS fragmentation, and effectively reduces sensitivity by splitting ion current. All of these factors deteriorate top down mass spectral data quality, an effect that becomes more pronounced as column diameter decreases. Artificial protein oxidation can also ...
Source: EuPA Open Proteomics - Category: Bioinformatics Source Type: research