Chemical derivatization of histones for facilitated analysis by mass spectrometry.
Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, we describe protocols for the characterization of histone modifications in both qualitative and semiquantitative manners using chemical derivatization and tandem mass spectrometry. In these procedures, extracted histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addition, a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodology has the advantage of producing a small number of predicted peptides from highly modified proteins. The protocol should take approximately 15-19 h to complete, including all chemical reactions, enzymatic digestion and mass spectrometry experiments.
Garcia
Benjamin A
BA
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Mollah
Sahana
S
Ueberheide
Beatrix M
BM
Busby
Scott A
SA
Muratore
Tara L
TL
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
D000595:Amino Acid Sequence; D006657:Histones / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006657:Histones