A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Abstract

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

Keywords: PARP14/ARTD8; inflammation; mass spectrometry; posttranslational modification; proteomics.

Graphical abstract

Fig. 1

ADP-ribosylome bench to spectral annotation workflow. A, IFN-γ induced ADP-ribosylome study. Ten-week old sibling mice were injected with saline or IFN-γ, or nothing at all (no treatment control) (n = 6 per treatment group). Spleen and liver organs were harvested for subsequent mono-ADPr (MAR)ylated peptide enrichment. The samples were analyzed by the Q Exactive and/or the Lumos, and the spectra were annotated using SEQUEST via Proteome Discoverer 2.4. B, the ADPr structure and fragment ions (modification ion, m-ion (and [MH]+ values); the complementary peptide ion, p-ion). Amino acid acceptor sites are indicated. C, schematic of a typical HCD-generated ADPr peptide spectrum. D, candidate ADPr spectra identified and scored (XCorr) by the search engine (SEQUEST-HT) are then supplemented with m-ion and p-ion series scores to help validate the assignments.

Fig. 2

Distributions of ADPr, non-ADPr, and unidentified spectra in collision energy experiments. A, the impact of varying instrument platform, dissociation method, and collision energy on the number of annotated ADPr spectra versus non-ADPr and unidentified spectra (individual numbers are labeled within each bar). The average collision energy percentages are plotted to the right. Annotated spectra include only rank1 peptides (5% FDR). B, the relationship between XCorr and collision energy (ColE) for ADPr and non-ADPr peptides. C–E, an ADPr peptide from mouse liver Selenium-binding protein 1/2 (SELENBP1/2) sequenced by HCD CE 28% (C) or CE 34% (D) and CID CE 40% (E) on the Lumos. The purple arrows highlight precursor P-ions that tend to predominate at lower HCD collision energies (e.g., CE 28%) or in CID spectra; but dissociate further as HCD collision energy increases (e.g., CE 34%). Fully annotated spectra are in supplemental Figure S4.

Fig. 3

ADPr peptide m-ion score distributions. A, the m-ion series score distributions calculated from the spectra of annotated ADPr and non-ADPr PSMs (Rank 1, 5% FDR). B, a closer analysis of the m-series score distributions comparing Lumos HCD (28% CE)-generated ADPr and non-ADPr PSMs. C, the m-series score distribution for the corresponding unidentified spectra from panel B. Candidate ADPr spectra still contained within the unidentified spectra are highlighted. D, distribution of the precursor mass ([MH]+) across the spectral categories. E, distribution of percent isolation interference across spectral categories. F, an example workflow strategy (i.e., semi-trypsin search) to further annotate ADPr spectra from the unidentified category. G, example semi-tryptic ADPr spectrum previously contained within unidentified (default fully tryptic search) spectral category.

Fig. 4

The m-ions undergo sequential dissociation. A, the m1/P10-ion dynamics with two HCD collision energy settings on the Lumos. B, the m6/P5-ion dynamics with two HCD collision energy settings on the Lumos. C and D, an ADPr peptide analyzed by CID (C) or HCD (D) on the Lumos, demonstrating that the ADPr molecule is more readily detected in CID but not HCD, likely due to sequential dissociation. E, the P1-ion is prevalent in HCD and CID scans, despite the low signal for the corresponding m10-ion in HCD data, suggesting the m10-ion dissociates further in HCD. F, A summary of the dissociation properties of ADPr peptides when using HCD.

Fig. 5

P-series support inclusion of medium confidence ADPr peptide identifications. A–C, p-series versus XCorr grouped by precursor charge. PSMs are rank 1, 5% FDR. XCorr thresholds (0.8, z = 2+; 1.0, z = 3+; 1.2, z > 3+) are based on the Fixed PSM Scorer's medium confidence thresholds. The p-series cut-off was based on manual inspection of several spectra. PSMs inside of gray area will not be considered further. D–F, example medium confidence (5% FDR), low XCorr ADPr spectra supported by p-series ions. MTRF1, Peptide chain release factor 1; P4HB, Protein disulfide-isomerase; PGM1, Phosphoglucomutase-1.

Fig. 6

The distinct ADP-ribosylomes from mouse liver and spleen. A, total Rank 1 unique ADPr peptide sequences identified using HCD and EThcD events triggered from the same precursor scan, and their corresponding proteins. B, overlap between liver and spleen proteomes and liver and spleen ADPr proteins with their respective proteomes. The proteomes were analyzed with a single HCD acquisition, whereas the ADP-ribosylomes were analyzed by multiple gas-phase separation (GPS) acquisitions. Proteins from each portion of the Venn diagram are in supplemental Table S4. C, total ADPr proteins reported. D, STRING database “full network” output for the liver unique ADPr proteins: 361/379 proteins in the database; highest confidence edge strength reported, 0.900. E, STRING database “full network” output for the spleen unique ADPr proteins: 43/45 proteins in the database; at least medium confidence edge strength reported, 0.400. F, STRING database “full network” output for the overlapping common ADPr proteins: 47/50 in the database; at least high confidence edge strength reported, 0.700. For C–E, disconnected nodes are hidden; and k-means clusters (n = 10 for liver; n = 3 for spleen; n = 2 for common proteins) were chosen manually after iterations of varying cluster number with subsequent Gene Ontology analysis. Dashed lines are interactions separated by clusters.

Fig. 7

IFN-γ induces distinct responses in mouse liver and spleen. A, changes to mice spleen and liver proteomes in response to IFN-γ. The average of n = 3 (spleen pools) and n = 6 (liver) for saline versus IFN-γ. B, cell and tissue-specific terms recognized by the Mouse Gene Atlas database for IFN-γ-induced proteome changes. C, heat maps of two-group comparisons of changing ADPr peptides (p < 0.05; q = 0.23 spleen and q = 0.62 liver). [Gene name and [amino acid position]. D, p1-ions are assumed to be y-ions by the search engine. Target MS2 (tMS2) of a candidate His-modified ADPr confirms the downstream Ser to be the correct site. E, a p1-ion is assumed to be a b-ion by the search engine. The p-series annotation indicates the lower ranking (rank 2) peptide to be correct; EthcD supports the rank2 assignment. F, EThcD-based summary of ADPr acceptor sites. Only the GPS scan m/z 400–1500 was used for the acceptor site consensus. The highest scoring amino acid site for a given peptide sequence is reported.