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. 2016 Nov;15(11):3388-3404.
doi: 10.1074/mcp.M116.058073. Epub 2016 Aug 23.

Abundant Lysine Methylation and N-Terminal Acetylation in Sulfolobus islandicus Revealed by Bottom-Up and Top-Down Proteomics

Egor A Vorontsov  1 , Elena Rensen  2 , David Prangishvili  2 , Mart Krupovic  3 , Julia Chamot-Rooke  4   5
Affiliations

Abundant Lysine Methylation and N-Terminal Acetylation in Sulfolobus islandicus Revealed by Bottom-Up and Top-Down Proteomics

Egor A Vorontsov et al. Mol Cell Proteomics. 2016 Nov.

Abstract

Protein post-translational methylation has been reported to occur in archaea, including members of the genus Sulfolobus, but has never been characterized on a proteome-wide scale. Among important Sulfolobus proteins carrying such modification are the chromatin proteins that have been described to be methylated on lysine side chains, resembling eukaryotic histones in that aspect. To get more insight into the extent of this modification and its dynamics during the different growth steps of the thermoacidophylic archaeon S. islandicus LAL14/1, we performed a global and deep proteomic analysis using a combination of high-throughput bottom-up and top-down approaches on a single high-resolution mass spectrometer. 1,931 methylation sites on 751 proteins were found by the bottom-up analysis, with methylation sites on 526 proteins monitored throughout three cell culture growth stages: early-exponential, mid-exponential, and stationary. The top-down analysis revealed 3,978 proteoforms arising from 681 proteins, including 292 methylated proteoforms, 85 of which were comprehensively characterized. Methylated proteoforms of the five chromatin proteins (Alba1, Alba2, Cren7, Sul7d1, Sul7d2) were fully characterized by a combination of bottom-up and top-down data. The top-down analysis also revealed an increase of methylation during cell growth for two chromatin proteins, which had not been evidenced by bottom-up. These results shed new light on the ubiquitous lysine methylation throughout the S. islandicus proteome. Furthermore, we found that S. islandicus proteins are frequently acetylated at the N terminus, following the removal of the N-terminal methionine. This study highlights the great value of combining bottom-up and top-down proteomics for obtaining an unprecedented level of accuracy in detecting differentially modified intact proteoforms. The data have been deposited to the ProteomeXchange with identifiers PXD003074 and PXD004179.

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Figures

Fig. 1.
Fig. 1.
LFQ-based volcano plots showing the protein expression level changes between the growth stages of S. islandicus. A, Early to Mid stage, B, Mid to Late stage, C, Early to Late stage. Black solid lines represent the significance threshold at FDR 0.01 and S0 = 1. Color-coded functions are shown for some of the differentially expressed protein functional groups. Four DNA-binding proteins are also highlighted in red: Alba1, Alba2, proteins Sul7d1 and Sul7d2 (quantified together), and Cren7 protein.
Fig. 2.
Fig. 2.
A and B - representative spectra of the methylated peptides from S. islandicus Lys-C/trypsin digest, corresponding fragmentation maps are shown below the spectra. A, spectrum (ETD) contains the continuous ion series on both sides of the methylated lysine residue, thus the methylated lysine site is filtered in; B, (HCD) fragment ion series is discontinuous, hence methylated lysine residue discarded; C, Venn diagrams show the number of the found methylation sites per growth stage (circles are given for each biological replicate B.R. at the corresponding growth stage); D, Venn diagram, showing the combined number of sites and methylated proteins for the three growth stages, and well as for the SCX fractionated late growth stage sample; E, volcano plot of the normalized peptide intensity changes between Early and Late stages. Significantly changing K-Me peptides are show in red, significance threshold at FDR 0.05 and S0 = 2; F, sequence logos, showing the frequency of the amino acid residue occurrences in the proximity of a methylated lysine (left) or in proximity to any lysine residue (right).
Fig. 3.
Fig. 3.
Functional annotation of the identified methylated proteins. Pie-chart on the left shows the classification of identified methylated proteins according to functional categories, as defined in the archaeal clusters of orthologous groups (arCOG) (46, 47). The numbers in parenthesis indicate the exact number of identified methylated proteins in each category. The categories are denoted by capital letters and the key is provided in the right panel. Horizontal bars show the proportion of methylated proteins among all detected proteins in each functional category.
Fig. 4.
Fig. 4.
Summary of the top-down LC-MS/MS analysis of the SAX-prefractionated S. islandicus cytosolic sample: A, distributions of the experimental intact protein masses by fraction; B, numbers of protein IDs, identified in each SAX fraction, including a number of the unique IDs, found exclusively in the respective fraction, and nonunique IDs, which were also found in one or more other fractions; C, distribution of the proteoforms found per protein ID; D, distribution of the most frequent types of identified proteoforms. Fl, flow-through fraction.
Fig. 5.
Fig. 5.
Fragmentation maps, showing localization of methylations and acetylations on the main S. islandicus methylated chromatin protein forms by ETD or HCD MS/MS. A, Alba1 proteoforms; B, Alba2 proteoforms; C, Cren7 proteoforms; D, Sul7d1 proteoforms, E, Sul7d2 proteoforms. Red box represents N-terminal acetylation; green box, lysine methylation; light blue box, lysine dimethylation; violet box, lysine trimethylation; gray box, methionine monooxidation.
Fig. 6.
Fig. 6.
Relative quantification of Alba1 and Cren7 proteoforms by top-down experiments. A, averaged spectra of Alba1 proteoforms at three growth stages; B, averaged spectra of Cren7 proteoforms at three growth stages; C, relative abundances of the Alba1 methylated forms at three growth stages; D, relative abundances of the Cren7 methylated forms at three growth stages. Peak areas on the extracted ion chromatograms (XIC) for each form are related to XIC peak area of nonmethylated form in the corresponding chromatogram.
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