Fishing for newly synthesized proteins with phosphonate-handles

Abstract

Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term PhosID. In this approach, click-able phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.

Fig. 1. Phosphonic acid labeling of biomolecules and enrichment strategy.

a Scheme of CuAAC and SPAAC bioorthogonal reactions used in this study. b Synthetic route of the three different phosphonate-labeled probes. c General workflow for the phosphonate enrichment strategy (PhosID). Detailed experimental procedures and conditions are described in the “Methods” section.

Fig. 2. Labeling and enrichment of functionalized BSA by PhosID.

a Efficient PhosID enrichment as quantified by relative MS intensities. BSA was functionalized at the free amine groups of lysines to yield azide-modified BSA (BSA-N₃) or alkyne-modified BSA (BSA-N₃) and was clicked to the corresponding P-alkyne/P-DBCO or P-azide, respectively. After trypsin digestion, phosphonate-modified peptides were retrieved by IMAC purification. Relative intensity of BSA peptides detected in IMAC input, flowthrough (FT) and elution (enriched) fractions are plotted. Data from the control experiments are shown in Supplementary Fig. 2. b Sensitive retrieval of phosphonate-modified BSA peptides from complex Hela lysate. Digested BSA-N₃ was spiked in 100 µg of Hela digest in various proportions, clicked with P-alkyne and enriched by IMAC in the PhosID workflow as shown in Fig. 1c. The summed intensity of phosphonate-modified peptides are plotted relative to total intensity of all peptides detected in IMAC eluate (left axis). Green bars represent the number of unique phosphonate-labeled BSA peptides retrieved (right axis). Even at a BSA-N₃: HeLa ratio of 0.01:100 (ratio of 1:10,000; w/w), phosphonate-labeled BSA peptides were still well detectable. Source data provided in Source data file.

Fig. 3. Enrichment of AHA-labeled proteins from HeLa cell by PhosID or biotin-streptavidin workflow.

a Parallel steps in PhosID and biotin-streptavidin workflows. To make comparisons between these two workflows, HeLa cells were metabolically labeled with AHA for 24 h (pulse) or for about 3 weeks (stable), and half of the material was clicked to either P-alkyne or biotin-alkyne, for parallel comparisons. Enrichment via PhosID was performed at the peptide level as described herein, while enrichment of biotinylated proteins via streptavidin capture was performed at the protein level. b Relative MS intensities of phosphonate-modified or biotinylated peptides. PhosID was 95% selective for phosphonate-labeled peptides that also contained Met → AHA substitutions. Digestion of biotin-streptavidin enriched proteins on the other hand recovered only about 1% of peptides still tagged with biotin. c Comparison of identifications from PhosID and biotin-streptavidin workflows. PhosID identified only phosphonate-modified peptides with extremely low background, whereas the biotin-streptavidin approach picked up high background even in HeLa cells not labeled with AHA. Data based on three experimental replicates. A peptide or protein was considered valid when identified in at least two out of three replicates. Protein ID information is provided in Supplementary Data 1. d MS intensity correlation between pulse and stable AHA labeling. The extent of POI retrieval from pulse and stable AHA-labeled material were highly similar in both the PhosID and biotin-streptavidin approaches, suggesting that a short pulse of AHA is sufficient to profile the newly synthesized proteome sensitively. R² values based on the Pearson linear regression model reported. e Protein identification overlap between PhosID and Biotin-streptavidin workflows. Data based on 3 experimental replicates. A peptide or protein was considered valid if identified in at least two out of three replicates. Protein ID information is provided in Supplementary Data 3.

Fig. 4. Interferon gamma (IFNγ) responsive protein synthesis in HeLa cells.

a IFNγ stimulation timeline. NSPs induced by IFNγ were AHA-labeled, clicked to P-alkyne, and enriched using the PhosID protocol. Following IFNγ treatment for either 4 h or 24 h, the abundances of NSPs were determined and normalized against the respective untreated controls at the same time points. b Induction of IRF1 in IFNγ treated HeLa cells. Western blot shows a rapid induction of IRF1 at 4 h. By 24 h, IRF1 returned to pre-stimulation levels although downstream signaling and transcription events may still be active. Western blot source data provided in Source data file. c Volcano plot of IFNγ-responsive changes in the newly synthesized proteome. Protein targets in red are significantly induced by IFNγ stimulation by 2-, 5- or 10-fold in 24 h, while synthesis of protein targets in blue is significantly suppressed by IFNγ treatment in 24 h. A full list of differentially synthesized proteins is provided in the Supplementary Data 4. Data based on three experimental replicates. A protein was only quantified if identified in at least two out of three replicates.

Fig. 5. Comparison of IFNγ-responsive protein synthesis in HeLa and Jurkat cells.

a Heatmap of temporal IFNγ response in HeLa cells. As evident from the distinct red clusters of proteins, protein synthesis in HeLa cells followed, in general, a two-step induction. Fold change was calculated by the LFQ intensity ratio of IFNγ treated: control samples measured at each time. Data based on three experimental replicates. A protein was only quantified if identified in at least two out of three replicates. b Heatmap of temporal IFNγ response in Jurkat cells. As evident from the distinct red clusters of proteins, protein synthesis in HeLa cells followed, in general, a two-step induction. Fold change was calculated by the LFQ intensity ratio of IFNγ treated: control samples measured at each time. Data based on three experimental replicates. A protein was only quantified if identified in at least two out of three replicates. c Schematic summary of major temporal differences in IFNγ response. Network relationships were retrieved from STRING.