Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Abstract

Ribosomes are abundant, large RNA-protein complexes that are the sites of all protein synthesis in cells. Defects in ribosomal proteins (RPs), including proteoforms arising from genetic variations, alternative splicing of RNA transcripts, post-translational modifications and alterations of protein expression level, have been linked to a diverse range of diseases, including cancer and aging. Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets. In the present work, using E. coli 70S RPs as an example, we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry (MS) system, and then applied it to the HeLa 80S ribosome. The results were complemented by a bottom-up approach. In total, 50 out of 55 RPs were identified using the top-down approach. Among these, more than 30 RPs were found to have their N-terminal methionine removed. Additional modifications such as methylation, acetylation, and hydroxylation were also observed, and the modification sites were identified by bottom-up MS. In a HeLa 80S ribosomal sample, we identified 98 ribosomal proteoforms, among which multiple truncated 80S ribosomal proteoforms were observed, the type of information which is often overlooked by bottom-up experiments. Although their relevance to diseases is not yet known, the integration of top-down and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

Keywords: Bottom-up MS; Proteoforms; Ribosomal proteins; Top-down MS.

Graphical abstract

Fig. 1

Parameters optimized for top-down proteomics using quadrupole time-of-flight (Q-TOF) mass spectrometry. (A) Scan time vs. number of protein identification. (B) and (C) Number of proteins identified under different collision energy conditions. LME: low mass collision ramp energy; HME: high mass collision ramp energy.

Fig. 2

Top-down and bottom-up proteomics characterization of E.coli ribosome. (A) Overlaid base peak chromatograms of three technical replicates from E.coli ribosome. (B) The number of proteins identified in 30S and 50S using top-down and bottom-up approach, respectively. (C) Venn diagram showing the overlap of top-down and bottom-up identification.

Fig. 3

Top-down and bottom-up characterization of L12 and L7. (A) Base peak chromatograms of L12 and L7. (B) Corresponding mass spectrometry (MS) spectra of L7 and L12. (C) Expanded MS spectra of L12 and L7. Sequence maps of (D) L7 and (E) L12 proteoforms, respectively. (F) MS/MS spectrum of peptide at m/z 677.8559.

Fig. 4

Top-down mass spectrometry (MS) spectra and sequence coverage of 50S ribosomal protein (RP) L24 and truncated L24 (amino acids (AAs) 55−104): (A) MS spectrum. (B) and (D) Sequence map and MS/MS spectrum of 50S RP L24. (C) and (E) Sequence map and MS/MS spectrum of truncated 50S RP L24 (AAs 55−104).

Fig. 5

HeLa 80S ribosome structure and ribosomal proteoforms identified using a top-down proteomics approach. (A) HeLa 80S ribosomal proteoforms identified using a top-down proteomics approach. (B) 60S RP L19 structure under pre- and post-translocation. (C) Observed ribosomal proteoforms are highlighted on the structure of 80S ribosome. PTM: post-translational modification; DiMe: dimethylation; Ac: acetylation; Hydroxy: hydroxylation; Me: methylation; Met: methionine.