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Table 1 Summary of N-terminal tail modifications observed on H2A and H2A.X-F by mass spectrometry

From: Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development

A. H2A

   

Relative abundance on H2A

Peptide

Modifications

Sites

Egg

Pronuclei

S2 Cells

1-56 (GluC)

1 Phospho

S1

•••

•••••

••

1 Ac

K9/K5a

•

••

•••

1 Phospho + Ac

S1 + K9a

---

••

---

1-11 and 4–11 (Trypsin)

1 Me

R3

••

••

 

1 Ac

K9/K5a

••

••

 

B. H2A.X-F

 

Relative abundance on H2A.X-F

Peptide

Modifications

Sites

Egg

Pronoclei

H2A.X-F1

H2A.X-F2

H2A.X-F1

H2A.X-F2

1-31 (Chymotrypsin)

Unmodified (αN-Acetyl)

 

•••••

•••••

•••

••••

 

S1

•••

•••

••••

••••

 

R3

•••

•••

••

••

 

R3

••

••

•

•

 

K5a

•

••

••

••

1 Phospho + 1 Me

S1 + R3

••

••

•••

•••

1 Phospho + 2 Me

S1 + R3

•

•

••

••

1 Phospho + 1 Ac

S1 + K5a

---

---

••

••

  1. A. PTMs observed for the GluC-generated, 1–56 peptide and the tryptic peptides, 1–11 and 4–11 from canonical H2A samples. Note that in addition to the modified forms shown, unmodified and monomethylated forms were also detected (data not shown). We estimate that, taken together, unmodified (α-N-acetylated) and methylated H2A comprise approximately 80%, 20%, and 70% of the total canonical H2A in egg, pronuclei, and S3 cells, respectively. B. PTMs observed for the chymotrypsin generated, 1–31 peptide from H2A.X-F samples. Relative abundance values are reported as follows: −−− = not detected, • = 0.1- < 1%, •• = 1- < 10%, ••• = 10- < 25%, •••• = 25- < 50%, ••••• = ≥50%. Peptide forms that were observed at less than 0.1% relative abundance are not shown.
  2. aK-Ac could often be localized to more than one location. For canonical H2A, lysine acetylation was predominantly found at K9 in egg and pronuclei samples, with some K5ac present. In S3 cells, K5ac was most abundant with some K9ac. In H2A.X-F samples across all cell types, the acetylation was primarily observed at K5 with some K6ac (K6ac approximately 10 to 100 times less abundant than K5ac). These peptides were confirmed with MS/MS and accurate mass analysis. To determine the relative abundances of each modified form of the peptide, the areas under the extracted ion chromatogram for each peptide in all of its charge states and modified forms were integrated and summed. The peak area for each modified form was divided by the total area for all unmodified and modified forms present for each peptide to obtain an estimate of the percent of each form of modified peptide.