Therefore, the PGC column allows the quick separation of histone peptides, in MS compatible buffers, and removes salts from the sample, thereby reducing sample loss, the effects of ion suppression and improving dynamic range while not increasing the amount of downstream MS and data analysis meaning that the method is still amenable to high throughput methodologies. Conclusions The offline HPLC fractionation of peptides generated from the bottom up analysis of acid extracted histone proteins was performed using a PGC based hypercarb column in the first dimension, in conjunction with LC-MS analysis using C18 stationary phase in the second dimension. This first dimension separation uses typical reverse phase solvents that are readily compatible with the second dimension following removal of the acetonitrile and shows a high degree of orthogonality to C18 RP separations. Using this approach up to a 62.7% increase in histone PTMs was detected and quantified compared to traditional 1D-LC analysis on the same MS instrument. This method enabled more low abundant peptides to be identified and reliably quantified from an increased dynamic range. This method is an improvement on previous SCX based 2D-LC methodologies in terms of throughput and reduction in sample loss as no desalting or buffer exchange of the sample is required prior to LC-MS analysis. The major downside to using a 2D-LC-MS/MS approach is the increased time for both the MS acquisition and data processing compared to standard shotgun approaches. To combat the latter of these two issues a Histomatic script was developed. The use of the Histomatic allows the rapid, automated, high throughput processing of data generated from 2D-LC-MS/MS experiments.
Therefore, the PGC column allows the quick separation of histone peptides, in MS compatible buffers, and removes salts from the sample, thereby reducing sample loss, the effects of ion suppression and improving dynamic range while not increasing the amount of downstream MS and data analysis meaning that the method is still amenable to high throughput methodologies. Conclusions The offline HPLC fractionation of peptides generated from the bottom up analysis of acid extracted histone proteins was performed using a PGC based hypercarb column in the first dimension, in conjunction with LC-MS analysis using C18 stationary phase in the second dimension. This first dimension separation uses typical reverse phase solvents that are readily compatible with the second dimension following removal of the acetonitrile and shows a high degree of orthogonality to C18 RP separations. Using this approach up to a 62.7% increase in histone PTMs was detected and quantified compared to traditional 1D-LC analysis on the same MS instrument. This method enabled more low abundant peptides to be identified and reliably quantified from an increased dynamic range. This method is an improvement on previous SCX based 2D-LC methodologies in terms of throughput and reduction in sample loss as no desalting or buffer exchange of the sample is required prior to LC-MS analysis. The major downside to using a 2D-LC-MS/MS approach is the increased time for both the MS acquisition and data processing compared to standard shotgun approaches. To combat the latter of these two issues a Histomatic script was developed. The use of the Histomatic allows the rapid, automated, high throughput processing of data generated from 2D-LC-MS/MS experiments.
Tom%20Minshull%20Thesis%20.docx