Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation.

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms.

Profiling the Human Phosphoproteome to Estimate the True Extent of Protein Phosphorylation.

Public phosphorylation databases such as PhosphoSitePlus (PSP) and PeptideAtlas (PA) compile results from published papers or openly available mass spectrometry (MS) data. However, there is no database-level control for false discovery of sites, likely leading to the overestimation of true phosphosites. By profiling the human phosphoproteome, we estimate the false discovery rate (FDR) of phosphosites and predict a more realistic count of true identifications. We rank sites into phosphorylation likelihood sets and analyze them in terms of conservation across 100 species, sequence properties, and functional annotations. We demonstrate significant differences between the sets and develop a method for independent phosphosite FDR estimation. Remarkably, we report estimated FDRs of 84, 98, and 82% within sets of phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) sites, respectively, that are supported by only a single piece of identification evidence─the majority of sites in PSP. We estimate that around 62 000 Ser, 8000 Thr, and 12 000 Tyr phosphosites in the human proteome are likely to be true, which is lower than most published estimates. Furthermore, our analysis estimates that 86 000 Ser, 50 000 Thr, and 26 000 Tyr phosphosites are likely false-positive identifications, highlighting the significant potential of false-positive data to be present in phosphorylation databases.

Evaluation of the aMAP score for hepatocellular carcinoma surveillance: a realistic opportunity to risk stratify.

BACKGROUND AND AIMS: The aMAP score is a model that predicts risk of hepatocellular carcinoma (HCC) development in patients with chronic hepatitis. Its performance in a 'real world' surveillance setting has not yet been ascertained. PATIENTS AND METHODS: We had access to a cohort of 3473 individuals enrolled in a rigorously implemented and prospectively accrued surveillance programme (patients undergoing regular ultrasound and biomarker examination between 1998 and 2021). During this period 445 had HCC detected. Of these, 77.8% had early stage disease (within Milan criteria), permitting potentially curative therapy to be implemented in nearly 70% of cases. We applied the recently developed aMAP score to classify patients according to their initial aMAP score in to low, medium and high-risk groups as proposed in the original publication. The performance of the aMAP score was assessed according to the concordance-index and calibration (i.e. agreement between observed and predicted risk). Allowance was made for competing causes of death. RESULTS: The aMAP score achieved an overall C-index of 0.81 (95% CI: 0.79-0.82) consistent with the initial report and was unaffected by allowance for competing causes of death. Sub-group analysis showed that the results did not change significantly according to gender, or aetiology. However, aMAP discrimination was greater for younger individuals (versus older individuals), and also for individuals without cirrhosis. The HCC incidence rate was 0.98, 7.05 and 29.1 events per 1000 person-years in the low-, moderate- and high-risk aMAP groups, respectively. CONCLUSIONS: The results from this 'real-world' cohort demonstrate that risk stratification is a realistic prospect and that identification of a subgroup of chronic liver disease patients who have a very low risk of HCC is feasible.

Temporal modulation of the NF-κB RelA network in response to different types of DNA damage.

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics.

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely 'non-canonical' substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.