On the acquisition of +1 charge states during high-throughput proteomics: Implications on reproducibility, number and confidence of protein identifications

Abstract

Modern high-throughput methods for the proteome analysis are gradually replacing more traditional 2D gel-based techniques. Almost immediately after the introduction of high-throughput proteomics techniques in 2001, reproducibility of the results became an issue. Extensive discussion in the literature led to the conclusion that certain "undersampling" exhibited during measurements could be due to the stochastic nature of the data-dependent sampling, routinely used with current mass spectrometry equipment. At the same time, the effect of the acquisition of different charge states on the reproducibility and confidence of protein identifications, to the best of our knowledge, has never been properly evaluated. There exists the frequently voiced yet hardly documented opinion that +1 charge states should be rejected during data-dependent acquisition. The work presented here shows that inclusion of the +1 charge state in the data-dependent acquisition protocols can indeed lead to improved proteome coverage, reproducibility, and the confidence of protein identifications by high-throughput proteomics. It was also shown that contrary to the established opinion, gas-phase dissociation of singly charged peptide species results in rich fragmentation patterns containing both b- and y-ions allowing for successful and confident peptide identification.