ATS - American Thoracic Society BPC- base peak chromatogram BSA – Bovine serum albumin CARS- Compensatory anti-inflammatory response syndrome ChAP-MS - chromatin affinity purification with mass spectrometry ChIP - chromatin immunoprecipitation ChIP-seq - chromatin immunoprecipitation - sequencing CID - collision induced dissociation - citrullination of histone H3 CLP – cecal ligature and puncture DA – Data analysis DC - direct current DIC - disseminated intravascular coagulopathy ECD - electron capture dissociation ELISA - Enzyme linked immunosorbant assay ESI - electrospray ionization ESICM - European Society of Intensive Care Medicine ETD - electron transfer dissociation FCS- Fetal calf serum FT-ICR - fourier transform ion cyclotron resonance G-CSF- granulocyte colony stimulating factor HAT - histone acetyl transferase HAT- Histone acetylase HDAC- Histone deactylase HILIC - Hydrophilic interaction chromatography HKMT- histone lysine methyltransferase HMT - histone methyl transferase enzyme HPLC - high performance liquid chromatography system HRP – Horseradish peroxidase ICU – intensive care unit IL-1 Interlukin-1 K- lysine KMT - lysine methyltransferases LC – Liquid chromatography LC-MS/MS - liquid chromatography – tandem mass spectrometry LPS – lipopolysaccharide LPS – Lipopolysaccharide MALDI - matrix assisted laser desorption ionization MAP- Mean Arterial Pressure MDMs – Monocyte derived macrophages Me1- monomethylation Me2- dimethylation Me3- trimethylation MHC – histocompatibility complex MILP - integer linear optimization MS - mass spectrometry MS/MS – tandem mass spectrometry N – Asparagine NETs - neutrophil extracellular traps NT- non tolerisable genes OP- over propionylation PADI - peptidyl arginine deiminase PAMP - pathogen associated molecular pattern PBMCs – Peripheral blood mononuclear cells
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.
(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.