Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: an application for determining lysine 9 acetylation and methylation of histone H3.

Histone acetylation and methylation play a critical role in transcription and gene regulation. Identification of sites of lysine acetylation and methylation in histones or other proteins by mass spectrometry (MS) is of increasing interest. In this paper, we report the use of MS to differentiate between peptides containing acetylated or tri-methylated lysines. High accuracy matrix-assisted laser desorption/ionization-time of flight MS gives better than five parts per million measurement accuracy, which is sufficient to verify acetylation and/or methylation. Electrospray ionization tandem mass spectrometry was used to assign modification sites and to differentiate acetylation from methylation. Typically, an immonium ion at m/z 98 corresponds to a mono-methylated lysine and an immonium ion at m/z 126 corresponds to an acetylated lysine. The neutral loss ion (MH(+)-59) is unique for a tri-methylated lysine. For a peptide with two or more modification sites of acetylation or tri-methylation or one site containing partial acetylation and tri-methylation, the a(2)-, b(2)-type ion is the characteristic index for an acetylated lysine whereas the b(2)-59 ion is indicative of a tri-methylated lysine in the N-terminus. The y-type ions and y-59 ions are characteristic of an acetylated lysine and a tri-methylated lysine at the C-terminus, respectively. We demonstrated that a lysine in a peptide modified by methylation or acetylation can be differentiated by MS using our method. Even if more then one lysine is present in a peptide and different modifications of this amino acid occur, they can be distinguished. This method was successful for the determination of the acetylation and methylation status of lysine 9 of histone H3 in chicken erythrocytes and human HeLa cell lines.

Cell shape change precedes staurosporine-induced stabilization and accumulation of p27kip1.

The requirement of an intact cytoskeleton organization for G1/S cell cycle progression has been demonstrated in cultured cells. In the non-small-cell lung carcinoma cell line A549, the kinase inhibitor staurosporine induced G1 cell cycle arrest with an accumulation of the cyclin-dependent kinase inhibitor p27kip1. Staurosporine induced also a drastic change in cell shape that was accompanied by changes in the actin cytoskeleton. The cytoskeleton disruption agents, cytochalasin D (cyto D) and 2,3-butanedione 2-monoxime (BDM), also induced G1 cell cycle arrest in A549 cells but without an accumulation of p27kip1. A comparison of the cell shape changes caused by these agents revealed that a conversion from an epithelial polygonal shape to an elongated fibroblast-like shape was specific for staurosporine. The shape change induced by staurosporine preceded the accumulation of p27kip1 by about 4 h. The accumulation of p27kip1 was not due to enhanced transcription but to stabilization of the protein resulting from the inhibition of proteolytic degradation. Staurosporine, however, did not inhibit directly the proteasome that was involved in the cell-cycle-dependent p27kip1 degradation. The results indicate that the cell shape change caused by staurosporine correlates with the accumulation of p27kip1 and that staurosporine interferes with the p27kip1-specific proteolysis activity.