Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a

Abstract

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.

Figure 1

Differentiating post-translational modification isomers by tandem mass spectrometry. a. CAD tandem spectrum of an [M+2H]2+ ion at 925.535 m/z at from the propionylated HMGA1a trypsin digest showing the peptide with the sequence _pr-GRPK_prGSK_prNK_prGAAK_me1TR (Lys70me1). b. CAD MS/MS spectrum and fragment map for a doubly charged ion at the same 925.535 m/z as the species in (a.).Nevertheless, the b and y type fragments allow for the identification of two different distinct species having the sequences of the HMGA1a peptide _pr -GRPK_prme1GSK_pr NK_pr GAAK_pr TR (Lys61me1) and_pr -GRPK_prGSK_prme1NK_pr GAAK_pr TR (Lys64me1), pr= propionyl amide, ac = acetyl.

Figure 2

Middle Down MS analysis of HMGA1a. ETD MS/MS spectrum of a triply charged ion at 680.335 m/z from the limited trypsin digest of HMGA1a. This peptide was determined to primarily be KQPPVSphosPGTALVGSQKEPSEVPTphosPK, displaying a combinatorial phosphorylation pattern.

Figure 3

Sequence coverage and post-translational modifications found on HMGA1a through nanoLC-MS/MS approaches (Bottom Up and Middle Down). Sequence coverage is indicated by underlined regions.

Figure 4

Full mass spectrum of the 14^th charge state of HMGA1A derived from HeLa S3 cells grown in suspension. The modification state of the peaks are labeled based on accurate mass analysis (see Table 2) and Top Down ETD tandem MS sequencing (see Figure 7). Note that the spectrum is dominated by the diphosphorylated species. Note also that the relative ratio within a phosphorylation state of the methylated to unmethylated forms is consistent across different degrees of phosphorylation.

Figure 5

A Top Down ETD spectrum of the diphosphorylated forms of HMGA1A isolated at 841.5 m/z ± 1.5 m/z. The major forms are identified are both N-terminally acetylated and diphosphorylated near the C-terminus and the less abundant form is monomethylated at Arg25. The spectrum is broken into four unequal sections for clarity. Due to space limitations not all identified peaks are labeled.

Figure 6

ETD based fragment map of the Top Down MS analysis of HMGA1a obtained from fragmentation produce in Figure 7. The methylation on Arg25 is variable (there is also an unmethylated form in the spectrum). The C-terminal phosphorylations are at two out of three of the serines indicated. The bottom up data only supports the two phosphorylations being at the two more C-terminal sites (Ser101phSer102ph).