Histone 4

We compared the performance of MS Amanda based on four data sets: an HCD HeLa sample, a synthetic peptide library, a histone data set, and a CID HeLa sample. The HCD HeLa sample, published by Michalski et al.,^{30 (link)} consists of three replicate measurements of tryptic peptides derived from one human cancer cell line. The synthetic peptide library, as described by Marx et al.,^{31 (link)} is composed of more than 200,000 phosphorylated and nonphosphorylated peptides. Performance comparisons were based on provided HCD and ETD data. The histone data set is composed of four different preparations, namely, Histone II-A from calf thymus (Sigma), Histone III-S from calf thymus (Sigma), Histone IV from Xenopus laevis, recombinantly expressed in Escherichia coli (Upstate), and Core Histones from chicken erythrocytes (Millipore). The published CID HeLa sample^{32 (link)} covers three replicates measured with a 1 h gradient (1μg).

For worm samples, proteins were extracted as described previously^{13 (link)}. Western blotting was performed with antibodies against GFP (1:1000, Cat. 2956, CST), Actin (1:2000, Cat. A5441, Sigma), H3K18Ac (1:1000, Cat. 07-354, Merck), H3K27Ac (1:1000, Cat. ab4729, abcam), H3K9Ac (1:1000, Cat. 06-942, Merck), H3K4Ac (1:1000, Cat. Ab176799, abcam), Histone 3 (1:1000, Cat. 9715, CST), Tubulin (1:2000, Cat. T5168, Sigma), H3K27Me3 (1:1000, Cat. 07-449, millipore), H3K27Me2 (1:1000, Cat. ab24684, abcam), H3K27Me1 (1:1000, Cat. 07-448, millipore), H3K9Me1 (1:1000, Cat. 07-450, millipore), H3K4Me3 (1:1000, Cat. 07-473, millipore), Histone 4 (1:1000, Cat. sc-10810, Santa Cruz), H4K5Ac (1:1000, Cat. ab51997, abcam), β-amyloid 1–16 (6E10) (1:1000, Cat. 803001, BioLegend), HA-tag (1:2000, Cat. 3724, CST), Flag-tag (1:1000, Cat. F7425, Sigma), Myc-tag (1:2000, Cat. sc-40, Santa Cruz), GST-tag (1:1000, Cat. 2625, CST), AcK (1:1000, Cat. 9441, CST), AcK (1:1000, Cat. 9814, CST) and HRP-labelled anti-rabbit (Cat. 7074, CST) and anti-mouse (Cat. 7076, CST) secondary antibodies.