Examining histone posttranslational modification patterns by high-resolution mass spectrometry.
Histone variants and posttranslational modifications (PTMs) are essential for epigenetic regulation of transcriptional expression. Single and/or combinatorial PTMs of histones play important roles in development and disease formation. Mass spectrometry (MS) has been a powerful tool to study histone variants and PTMs, as it not only can identify novel PTMs but also can provide quantitative measurement of a spectrum of histone variants and PTMs in the same sample. In this chapter, we employ a combination of chemical derivation and high-resolution MS to identify and quantify multiple histone variants and PTMs. Histones are acid extracted and modified with propionyl groups and subsequently produces suitable sizes of fragments for MS analysis by trypsin digestion. The newly generated N-termini of histone peptides can be differentially marked by stable isotope labeling in a second reaction of propionylation, which enables direct comparison between two different samples in the following MS analysis.
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Shu
S
Lin
Benjamin A
BA
Garcia
D000595:Amino Acid Sequence; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007475:Ion Exchange Resins / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D053719:Tandem Mass Spectrometry; D046509:Tissue Culture Techniques; D014025:Titanium / Q000737:chemistry; D014238:Trichloroacetic Acid / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D006657:Histones; D007475:Ion Exchange Resins; D010446:Peptide Fragments; D010750:Phosphoproteins; C009495:titanium dioxide; D014238:Trichloroacetic Acid; D014025:Titanium; D014357:Trypsin