Collective Mass Spectrometry Approaches Reveal Broad and Combinatorial Modification of High Mobility Group Protein A1a

• Published in: Journal of the American Society for Mass Spectrometry Vol. 21; no. 6; pp. 960 - 970
• Main Authors: Young, Nicolas L., Plazas-Mayorca, Mariana D., DiMaggio, Peter A., Flaniken, Ian Z., Beltran, Andrea J., Mishra, Neeli, LeRoy, Gary, Floudas, Christodoulos A., Garcia, Benjamin A.
• Format: Journal Article
• Language: English
• Published: New York Elsevier Inc 01.06.2010; Springer-Verlag; Springer Nature B.V
• Subjects: Acetylation; Amino Acid Sequence; Analytical Chemistry; Apoptosis; Bioinformatics; Biotechnology; Chemistry; Chemistry and Materials Science; Combinatorial analysis; HeLa Cells; Histones; HMGA1a Protein - chemistry; HMGA1a Protein - metabolism; Humans; Liquid chromatography; Mass spectrometry; Methylation; Molecular Sequence Data; Nanostructure; Organic Chemistry; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Mapping - methods; Phosphorylation; Protein Processing, Post-Translational; Proteins; Proteomics; Proteomics - methods; Scientific imaging; Spectroscopy; HMGA Protein; Grow HeLa Cell; Propionic Anhydride; High Mobility Group; Mass Spectrometry Analysis
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2010.01.020

Transcriptional states are formed and maintained by the interaction and post-translational modification (PTM) of several chromatin proteins, such as histones and high mobility group (HMG) proteins. Among these, HMGA1a, a small heterochromatin-associated nuclear protein has been shown to be post-translationally modified, and some of these PTMs have been linked to apoptosis and cancer. In cancerous cells, HMGA1a PTMs differ between metastatic and nonmetastatic cells, suggesting the existence of an HMGA1a PTM code analogous to the “histone code.” In this study, we expand on current knowledge by comprehensively characterizing PTMs on HMGA1a purified from human cells using both nanoflow liquid chromatography collision activated dissociation mediated Bottom Up and electron-transfer dissociation facilitated middle and Top Down mass spectrometry (MS). We find HMGA1a to be pervasively modified with many types of modifications such as methylation, acetylation, and phosphorylation, including finding novel sites. While Bottom Up MS identified lower level modification sites, Top and Middle Down MS were utilized to identify the most commonly occurring combinatorially modified forms. Remarkably, although we identify several individual modification sites through our Bottom Up and Middle Down MS analyses, we find relatively few combinatorially modified forms dominate the population through Top Down proteomics. The main combinatorial PTMs we find through the Top Down approach are N-terminal acetylation, Arg25 methylation along with phosphorylation of the three most C-terminal serine residues in primarily a diphosphorylated form. This report presents one of the most detailed analyses of HMGA1a to date and illustrates the strength of using a combined MS effort. Combined CID and ETD based Bottom Up, Middle Down, and Top Down mass spectrometry of HMGA1a post-translational modification find few combinatorial modifications.