(K27me3) are all isobaric, they have the same nominal mass). In order to determine the correct modification and its site a combination of the retention time and MS/MS was used (Figure 4.4). In the 3+ charge state all three of the peptides monoisotopic peaks are 548.66 m/z. When an EIC is generated for this m/z it reveals three peaks (Figure 4.4A). In order to assign each peak to its corresponding proteoform, the MS/MS spectra were examined, as can be seen in Figure 4.4B the Kme2SAPATGGVKme1KPHR peptide contains diagnostic shift in b2 ions at 371 which is diagnostic of a dimethylation, and doesn’t not contain either any peaks corresponding to a methylation or trimethylation. Importantly this spectrum also contains a mass shifts at y5 that is indicative of a methylation at this position and not the adjacent lysine confirming that this species is indeed the Kme2SAPATGGVKme1KPHR. This pattern of elution is consistent, even though the exact retention time will shift as it is dependent on the gradient and can be used as additional evidence. The trimethylated species elutes from the C18 column first followed by the K27me2K36me1 and finally K27me1K36me3 (Figure 4.4A). Mass spectrometry not only allows a vast amount of histone PTMs to be discovered in all histone proteins within a single experiment, but also allows combinations of these modifications to be identified. However, as a bottom up approach was used, only short range modifications were identified, due to the relatively short size of peptides generated. Despite this, of the 54 modified H3 peptides discovered, 25 were the result of the addition of multiple chemical groups. The MS analysis (summarised in Figure 4.3) highlights the combinatorial complexity that exists on the N-terminal tails, something that although is widely known to exist within histone proteins has not before been described in human primary macrophages.
(K27me3) are all isobaric, they have the same nominal mass). In order to determine the correct modification and its site a combination of the retention time and MS/MS was used (Figure 4.4). In the 3+ charge state all three of the peptides monoisotopic peaks are 548.66 m/z. When an EIC is generated for this m/z it reveals three peaks (Figure 4.4A). In order to assign each peak to its corresponding proteoform, the MS/MS spectra were examined, as can be seen in Figure 4.4B the Kme2SAPATGGVKme1KPHR peptide contains diagnostic shift in b2 ions at 371 which is diagnostic of a dimethylation, and doesn’t not contain either any peaks corresponding to a methylation or trimethylation. Importantly this spectrum also contains a mass shifts at y5 that is indicative of a methylation at this position and not the adjacent lysine confirming that this species is indeed the Kme2SAPATGGVKme1KPHR. This pattern of elution is consistent, even though the exact retention time will shift as it is dependent on the gradient and can be used as additional evidence. The trimethylated species elutes from the C18 column first followed by the K27me2K36me1 and finally K27me1K36me3 (Figure 4.4A). Mass spectrometry not only allows a vast amount of histone PTMs to be discovered in all histone proteins within a single experiment, but also allows combinations of these modifications to be identified. However, as a bottom up approach was used, only short range modifications were identified, due to the relatively short size of peptides generated. Despite this, of the 54 modified H3 peptides discovered, 25 were the result of the addition of multiple chemical groups. The MS analysis (summarised in Figure 4.3) highlights the combinatorial complexity that exists on the N-terminal tails, something that although is widely known to exist within histone proteins has not before been described in human primary macrophages.
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