A Quantitative Study on the In Vitro and In Vivo Acetylation of High Mobility Group A1 Proteins

• Published in: Journal of the American Society for Mass Spectrometry Vol. 18; no. 9; pp. 1569 - 1578
• Main Authors: Zhang, Qingchun, Zhang, Kangling, Zou, Yan, Perna, Avi, Wang, Yinsheng
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.09.2007; Elsevier Science; Springer Nature B.V
• Subjects: Acetylation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cell Line, Tumor; Chromatin; Chromatography, High Pressure Liquid - methods; Fundamental and applied biological sciences. Psychology; Histone Acetyltransferases - chemistry; Histone Acetyltransferases - metabolism; HMGA1a Protein - chemistry; HMGA1a Protein - metabolism; HMGA1b Protein - chemistry; HMGA1b Protein - metabolism; Holoproteins; Humans; In vivo methods and tests; Lysine; Male; Mass spectrometry; Neoplasm Proteins - chemistry; Neoplasm Proteins - metabolism; Nuclear proteins; Peptides; Phosphorylation; Prostate cancer; Prostatic Neoplasms - chemistry; Prostatic Neoplasms - metabolism; Proteins; Proteins - chemistry; Proteins - metabolism; Residues; Selectivity; Spectrometry, Mass, Electrospray Ionization - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Human; Chemical analysis; Peptides; Nuclear protein HMG; Coupled method; HPLC chromatography; In vitro; Electrospray; Posttranslational modification; In vivo; Cell line; Analysis method; Peptide fragment; Mass spectrometry MS/MS; Acetylation; Recombinant protein; Prostate; Tumor cell; Quantitative analysis
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2007.05.020

High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64∼Lys-70 > Lys-66 > Lys-14∼Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70∼Lys-73 > Lys-64∼Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.