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The clinical success of covalent drugs such as sotorasib has renewed interest in covalency for rational drug design. The most rigorous potency metric for covalent modifiers is the second-order rate constant k inact /K I . However, existing methods for measuring k inact / K I are resource-intensive and involve complex data interpretation. We describe the diagonal dose-response time-course (dDRTC), an efficient mass spectrometry-based method for determining k inact / K I , enabling routine k inact / K I quantification earlier in programs and accelerating SAR interpretation for lead discovery. We apply dDRTC to a dozen covalent fragment and lead-like modifiers for three targets, KRAS G12C and two E3 ligase complexes. Kinetic simulations comparing a range of k inact and K I values establish recommended parameters for dDRTC and reveal that the approach is particularly suited for covalent fragments and leads. Our results demonstrate accurate determination of k inact / K I values across three orders of magnitude with eight-fold increased throughput, reduced protein consumption, and simplified data analysis. In this work, Jeon et al. introduce the diagonal dose-response time-course (dDRTC), a fast and operationally simple mass spectrometry-based method for determining the efficiency of covalent modification ( k inact / K I ) across a wide dynamic range.

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of candidate proteins in patients with DILI at onset (DO; n  = 133) and follow-up ( n  = 120), acute non-DILI at onset (NDO; n  = 63) and follow-up ( n  = 42), and healthy volunteers (HV; n  = 104). Area under the receiver operating characteristic curve (AUC) for cytoplasmic aconitate hydratase, argininosuccinate synthase, carbamoylphosphate synthase, fumarylacetoacetase, fructose-1,6-bisphosphatase 1 (FBP1) across cohorts achieved near complete separation (range: 0.94–0.99) of DO and HV. In addition, we show that FBP1, alone or in combination with glutathione S-transferase A1 and leukocyte cell-derived chemotaxin 2, could potentially assist in clinical diagnosis by distinguishing NDO from DO (AUC range: 0.65–0.78), but further technical and clinical validation of these candidate biomarkers is needed. Diagnosis of rare, unpredictable, drug-induced liver injury (DILI) is a significant challenge for patients, clinicians, and drug development. Here, the authors discover, evaluate, and validate potential blood biomarkers to diagnose DILI and distinguish it from alternative causes of liver injury.

Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7-15 Sec residues depending on species. Assessing an individual's selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys.

Kidney fibrosis represents an urgent unmet clinical need due to the lack of effective therapies and an inadequate understanding of the molecular pathogenesis. We have generated a comprehensive and combined multi-omics dataset (proteomics, mRNA and small RNA transcriptomics) of fibrotic kidneys that is searchable through a user-friendly web application: http://hbcreports.med.harvard.edu/fmm/. Two commonly used mouse models were utilized: a reversible chemical-induced injury model (folic acid (FA) induced nephropathy) and an irreversible surgically-induced fibrosis model (unilateral ureteral obstruction (UUO)). mRNA and small RNA sequencing, as well as 10-plex tandem mass tag (TMT) proteomics were performed with kidney samples from different time points over the course of fibrosis development. The bioinformatics workflow used to process, technically validate, and combine the single omics data will be described. In summary, we present temporal multi-omics data from fibrotic mouse kidneys that are accessible through an interrogation tool (Mouse Kidney Fibromics browser) to provide a searchable transcriptome and proteome for kidney fibrosis researchers.

Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro , SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects.

Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA [Ser]Sec and its insertion was dependent on the Sec insertion sequence element in the 3' UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA [Ser]Sec to generate Cys-tRNA [Ser]Sec . Cys was inserted in vivo at UGA condons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinery-based mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.

We performed quantitative proteomics on 60 human-derived breast cancer cell line models to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics.

The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or ‘phospho‐form’—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the ‘phospho‐form distribution’: the relative amount of each of the 2 n phospho‐forms of a protein with n ‐phosphorylation sites. We compared four potential methods—western blots with phospho‐specific antibodies, peptide‐based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein‐based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)—on differentially phosphorylated samples of the well‐studied mitogen‐activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16‐member phospho‐form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem. Synopsis Protein post‐translational modification is one of the most significant regulatory mechanisms in cellular physiology, and protein phosphorylation is the most widely studied of these. An individual molecule may be phosphorylatable on multiple residues, allowing it to exist in a multiplicity of combinatorial patterns of modification; with n ‐sites, there may be 2 n such ‘phospho‐forms’. Recent work on many distinct types of proteins—ion channels, signalling enzymes, transcription factors, co‐activators, circadian clock components—has shown that different phospho‐forms may have different downstream effects. The impact of multisite phosphorylation, therefore, is determined by the proportions of the various phospho‐forms that are present in the molecular population of the given protein. This ‘phospho‐form distribution’ is dynamically and collectively regulated by the opposing actions of the relevant kinases and phosphatases. This presents a more challenging perspective than is depicted in the typical cartoon diagram, in which is shown only a single phospho‐form, usually the maximally phosphorylated one, and the underlying dynamics of modification and demodification is left implicit. The present paper sets out to bring this perspective of phospho‐forms and phospho‐form distributions to a wider biological audience, to compare current methods for measuring them and to discuss the challenges in developing a general strategy applicable to the kinds of proteins typically found in cellular physiology. We chose to analyse the 42 kDa mitogen‐activated protein (MAP) kinase Erk2 (Erk). Erk is a paradigmatic signalling protein that is phosphorylated on T and Y residues in a TEY motif within its kinase‐activation loop. Because these sites are so close together, multiple methods may be used to detect phospho‐forms and we compared four: western blots with phospho‐specific antibodies; peptide‐based liquid chromatography (LC)/mass spectrometry (MS), in which proteins are first digested into peptide (pepMS); protein‐based LC/MS with intact proteins (proMS); and nuclear magnetic resonance spectroscopy (NMR). To provide a stringent comparison, we used specific kinases and phosphatases to prepare four samples of Erk in distinct states of phosphorylation and sought to determine the proportion of each of the four phospho‐forms in each of the four samples. We found excellent agreement, to within 10%, between the various biophysical methods, pepMS, proMS and NMR, despite the experiments being carried out in three different laboratories on two different continents. NMR also revealed the presence of two additional phosphorylations on one of the samples, which we identified by MS as serine phosphorylations. We determined most of the corresponding 16 member phospho‐form distribution using a combination of pepMS and proMS. To our surprise, however, we found significant semi‐quantitative discrepancies between the biophysical and the immunological methods, despite using the LICOR method of ratiometric fluorescent imaging for western blotting. For instance, a phospho‐specific antibody may indicate that sample one has a higher proportion of a certain phospho‐form than sample two, but pepMS measurements may sometimes indicate the reverse (compare Figures 1D and 2C ). We found similar discrepancies with an alternative set of samples, prepared differently (compare Figure 3A and B ), and after spiking western blots with whole‐cell lysate (Figure 3C ) and after using chemiluminescence and CCD imaging in place of fluorescent detection. Antibodies are usually characterised for the purposes of quantitative measurement by titration against the same sample, but molecular recognition between antibody and antigen is an emergent property of the biological context. In the comparisons made here, the same phospho‐form is being examined in different samples and hence in the context of different phospho‐form distributions. The antibody sees not only the phospho‐epitope that is its nominal target but also differing amounts of other phospho‐epitopes against which it may have a range of cross‐reactivities. Such a context‐dependent interaction may be one reason for the surprising discrepancies that were found. If so, it exemplifies one of our central themes: it is not any single phospho‐form that determines the downstream response, in this case of an antibody; it is the entire phospho‐form distribution. While antibodies remain unrivalled for protein detection sensitivity amidst complex cellular backgrounds, our results suggest that care is required in using them for quantitative comparisons of post‐translational modifications. If sites can be localised to a single peptide, pepMS with both LC and MS offers good opportunities for separating distinct phospho‐forms, including isobaric ones having the same molecular weight. However, such measurements are not quantitative because distinct phospho‐forms may ionise and ‘fly’ with different efficiencies; isotopically‐labelled phospho‐peptide internal standards are required for accurate measurements. Moreover, sites on distinct peptides can no longer be correlated with each other. Measurements with intact protein, as in proMS, avoid both problems but are also less sensitive to LC separation, allowing only the distribution of isobaric forms to be determined. The use of multiple samples, prepared with specific phosphatases, can also be informative. The combination of pepMS and proMS offers a hybrid strategy that represents a good balance between coverage and resolution and holds out promise as a general approach for proteins with small numbers of sites. NMR has certain inherent limitations, being unable to detect correlations between phosphorylations that are not close together and being better able to detect phosphorylation on serine and threonine than on tyrosine. However, it has the advantage, as does proMS, of not being biased by prior expectations as to where modifications are expected, illustrated by its uncovering of the two additional phosphorylations on Erk. While NMR's specialised requirements make it less feasible as a general methodology, it holds out the promise of real‐time measurements both in vitro and in intact cells. The problem of quantifying phospho‐form distribution remains very challenging as the number of phosphorylated sites increases but quantitative information is now becoming available for proteins such as Erk that are commonly encountered in cellular physiology. We compared four methods for quantifying the proportions of phospho‐forms, or ‘phospho‐form distribution’, of a multiply phosphorylated protein, using as an example the MAP kinase Erk2, with two principal phosphorylation sites. Measurements by mass spectrometry (MS) and by nuclear magnetic resonance (NMR) agreed to within 10%, but phospho‐specific antibody measurements exhibited semi‐quantitative discrepancies with these, sometimes suggesting reverse trends to those found by the biophysical methods. NMR revealed under our conditions that Erk was phosphorylated on four, not two, sites. A combination of peptide‐based MS and protein‐based MS provided an optimum strategy for determining the 16=2 4 member phospho‐form distribution.

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories. LC/QTOF MS combined with automated charge state deconvolution, spectral mirroring, and spectral subtraction were utilized to distinguish E. coli O157:H7 strains having identical clinical manifestation.