Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens.

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5-7. Here we describe a group of RNR proteins in Mollicutes-including Mycoplasma pathogens-that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR-some of which are developing resistance to antibiotics-are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (µ-oxo/µ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (µ-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a µ-oxo/µ-hydroxo-bridged MnIII/FeIII dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.

Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein.

Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving-Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C-H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apo-protein exhibiting greater MnII affinity than FeII affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C-H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.

Accumulation of endogenous peptides triggers a pathogen stress response in Arabidopsis thaliana.

The stepwise degradation of peptides to amino acids in plant mitochondria and chloroplasts is catalyzed by a network of oligopeptidases (presequence protease PreP, organellar oligopeptidase OOP) and aminopeptidases. In the present report, we show that the lack of oligopeptidase activity in Arabidopsis thaliana results in the accumulation of endogenous free peptides, mostly of chloroplastic origin (targeting peptides and degradation products). Using mRNA sequencing and deep coverage proteomics, allowing for the identification of 17 000 transcripts and 11 000 proteins, respectively, we uncover a peptide-stress response occurring in plants lacking PreP and OOP oligopeptidase activity. The peptide-stress response results in the activation of the classical plant defense pathways in the absence of pathogenic challenge. The constitutive activation of the pathogen-defense pathways imposes a strong growth penalty and a reduction of the plants reproductive fitness. Our results indicate that the absence of organellar oligopeptidases PreP1/2 and OOP results in the accumulation of peptides that are perceived as pathogenic effectors and activate the signaling pathways of plant-defense response.

A multi-step peptidolytic cascade for amino acid recovery in chloroplasts.

Plastids (including chloroplasts) are subcellular sites for a plethora of proteolytic reactions, required in functions ranging from protein biogenesis to quality control. Here we show that peptides generated from pre-protein maturation within chloroplasts of Arabidopsis thaliana are degraded to amino acids by a multi-step peptidolytic cascade consisting of oligopeptidases and aminopeptidases, effectively allowing the recovery of single amino acids within these organelles.

Ether cross-link formation in the R2-like ligand-binding oxidase.

R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.

HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics.

We present a liquid chromatography-mass spectrometry (LC-MS)-based method permitting unbiased (gene prediction-independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7-5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectively. The method also enabled deep proteome coverage, identifying 13,078 human and 10,637 mouse proteins.

Tartrate-resistant acid phosphatase (TRAP/ACP5) promotes metastasis-related properties via TGFß2/TßR and CD44 in MDA-MB-231 breast cancer cells.

BACKGROUND: Tartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness. METHODS: MDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes. RESULTS: We identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFß) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFß isoform 2 (TGFß2), TGFß receptor type 1 (TßR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical inhibition demonstrated that TRAP-dependent migration and proliferation is regulated via TGFß2/TßR, whereas proliferation beyond basal levels is regulated through CD44. CONCLUSION: Altogether, TRAP promotes metastasis-related cell properties in MDA-MB-231 breast cancer cells via TGFß2/TßR and CD44, thereby identifying a potential signaling mechanism associated to TRAP action in breast cancer cells.

Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor.

Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn(II) and Fe(II) in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189-17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.

Secretome protein signature of human gastrointestinal stromal tumor cells.

Strategies for correct diagnosis, treatment evaluation and recurrence prediction are important for the prognosis and mortality rates among cancer patients. In spite of major improvements in clinical management, gastrointestinal stromal tumors (GISTs) can still be deadly due to metastasis and recurrences, which confirms the unmet need of reliable follow-up modalities. Tumor-specific secreted, shed or leaked proteins (collectively known as secretome) are considered promising sources for biomarkers, and suitable for detection in biofluids. Herein, we stimulated cell secretion in the imatinib-sensitive GIST882 cell line and profiled the secretome, collected as conditioned media, by using a shotgun proteomics approach. We identified 764 proteins from all conditions combined, 51.3% being predicted as classically/non-classically secreted. The protein subsets found were dependent on the stimulatory condition. The significant increase in protein release by the classical pathway was strongly associated with markers already found in other cancer types. Furthermore, most of the released proteins were non-classically released and overlapped to a high degree with proteins of exosomal origin. Imatinib pre-treatment radically changed these secretory patterns, which can have clinical implications when investigating biomarkers in imatinib-treated versus non-treated GIST patients. Our results show, for the first time, that GISTs contain a secretome signature. In the search for suitable biomarkers in the more complex GIST patient samples, this study aids in the understanding of basic GIST secretome characteristics.

SpliceVista, a tool for splice variant identification and visualization in shotgun proteomics data.

Alternative splicing is a pervasive process in eukaryotic organisms. More than 90% of human genes have alternatively spliced products, and aberrant splicing has been shown to be associated with many diseases. Current methods employed in the detection of splice variants include prediction by clustering of expressed sequence tags, exon microarray, and mRNA sequencing, all methods focusing on RNA-level information. There is a lack of tools for analyzing splice variants at the protein level. Here, we present SpliceVista, a tool for splice variant identification and visualization based on mass spectrometry proteomics data. SpliceVista retrieves gene structure and translated sequences from alternative splicing databases and maps MS-identified peptides to splice variants. The visualization module plots the exon composition of each splice variant and aligns identified peptides with transcript positions. If quantitative mass spectrometry data are used, SpliceVista plots the quantitative patterns for each peptide and provides users with the option to cluster peptides based on their quantitative patterns. SpliceVista can identify splice-variant-specific peptides, providing the possibility for variant-specific analysis. The tool was tested on two experimental datasets (PXD000065 and PXD000134). In A431 cells treated with gefitinib, 2983 splice-variant-specific peptides corresponding to 939 splice variants were identified. Through comparison of splice-variant-centric, protein-centric, and gene-centric quantification, several genes (e.g. EIF4H) were found to have differentially regulated splice variants after gefitinib treatment. The same discrepancy between protein-centric and splice-centric quantification was detected in the other dataset, in which induced pluripotent stem cells were compared with parental fibroblast and human embryotic stem cells. In addition, SpliceVista can be used to visualize novel splice variants inferred from peptide-level evidence. In summary, SpliceVista enables visualization, detection, and differential quantification of protein splice variants that are often missed in current proteomics pipelines.

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein.

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn(II) < Fe(II) < Ni(II) < Co(II) < Cu(II) > Zn(II)). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn(II) and Fe(II) in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe(II) over Mn(II) as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.

Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts.

Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.

Defining, comparing, and improving iTRAQ quantification in mass spectrometry proteomics data.

The purpose of this study was to generate a basis for the decision of what protein quantities are reliable and find a way for accurate and precise protein quantification. To investigate this we have used thousands of peptide measurements to estimate variance and bias for quantification by iTRAQ (isobaric tags for relative and absolute quantification) mass spectrometry in complex human samples. A549 cell lysate was mixed in the proportions 2:2:1:1:2:2:1:1, fractionated by high resolution isoelectric focusing and liquid chromatography and analyzed by three mass spectrometry platforms; LTQ Orbitrap Velos, 4800 MALDI-TOF/TOF and 6530 Q-TOF. We have investigated how variance and bias in the iTRAQ reporter ions data are affected by common experimental variables such as sample amount, sample fractionation, fragmentation energy, and instrument platform. Based on this, we have suggested a concept for experimental design and a methodology for protein quantification. By using duplicate samples in each run, each experiment is validated based on its internal experimental variation. The duplicates are used for calculating peptide weights, unique to the experiment, which is used in the protein quantification. By weighting the peptides depending on reporter ion intensity, we can decrease the relative error in quantification at the protein level and assign a total weight to each protein that reflects the protein quantitation confidence. We also demonstrate the usability of this methodology in a cancer cell line experiment as well as in a clinical data set of lung cancer tissue samples. In conclusion, we have in this study developed a methodology for improved protein quantification in shotgun proteomics and introduced a way to assess quantification for proteins with few peptides. The experimental design and developed algorithms decreased the relative protein quantification error in the analysis of complex biological samples.

Enhanced information output from shotgun proteomics data by protein quantification and peptide quality control (PQPQ).

We present a tool to improve quantitative accuracy and precision in mass spectrometry based on shotgun proteomics: protein quantification by peptide quality control, PQPQ. The method is based on the assumption that the quantitative pattern of peptides derived from one protein will correlate over several samples. Dissonant patterns arise either from outlier peptides or because of the presence of different protein species. By correlation analysis, protein quantification by peptide quality control identifies and excludes outliers and detects the existence of different protein species. Alternative protein species are then quantified separately. By validating the algorithm on seven data sets related to different cancer studies we show that data processing by protein quantification by peptide quality control improves the information output from shotgun proteomics. Data from two labeling procedures and three different instrumental platforms was included in the evaluation. With this unique method using both peptide sequence data and quantitative data we can improve the quantitative accuracy and precision on the protein level and detect different protein species.

A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines.

Besides the isolation and identification of major histocompatibility complex I-restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective antitumor CD8+ T-cell-mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific antitumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate >8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and HEX software. The latter is a tool previously developed by Jacopo, 2020, able to identify tumor antigens similar to pathogen antigens in order to exploit molecular mimicry and tumor pathogen cross-reactive T cells in cancer vaccine development. The generated list of candidates (26 in total) was further tested in a functional characterization assay using interferon-γ enzyme-linked immunospot (ELISpot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, nontreated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach.

Therapeutic Cancer Vaccination with Immunopeptidomics-Discovered Antigens Confers Protective Antitumor Efficacy.

Knowledge of clinically targetable tumor antigens is becoming vital for broader design and utility of therapeutic cancer vaccines. This information is obtained reliably by directly interrogating the MHC-I presented peptide ligands, the immunopeptidome, with state-of-the-art mass spectrometry. Our manuscript describes direct identification of novel tumor antigens for an aggressive triple-negative breast cancer model. Immunopeptidome profiling revealed 2481 unique antigens, among them a novel ERV antigen originating from an endogenous retrovirus element. The clinical benefit and tumor control potential of the identified tumor antigens and ERV antigen were studied in a preclinical model using two vaccine platforms and therapeutic settings. Prominent control of established tumors was achieved using an oncolytic adenovirus platform designed for flexible and specific tumor targeting, namely PeptiCRAd. Our study presents a pipeline integrating immunopeptidome analysis-driven antigen discovery with a therapeutic cancer vaccine platform for improved personalized oncolytic immunotherapy.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry.

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Concentration-dependent front velocity of the autocatalytic hydrogenase reaction.

HynSL hydrogenase from Thiocapsa roseopersicina was applied to catalyze the oxidation of molecular hydrogen in a new, improved, thin-layer reaction chamber. Investigation of the nature of this catalysis via the development of reduced benzyl viologen showed clearly the typical characteristics of an autocatalytic reaction: propagation of a reaction front originating from a single point, with a constant velocity of front propagation. The dependence of the reaction velocity on enzyme concentration was a power function with a positive enzyme concentration threshold, with an exponent of 0.4 +/- 0.05. This indicates that the autocatalyst is an enzyme form. The front velocity decreased on increase of the electron acceptor concentration, as a sign that the autocatalyst interacts directly with the final electron acceptor. Overall, it may be concluded that the autocatalyst is an enzyme form in which [FeS]distal is reduced. Model calculations corroborate this. Because the reduction of all [FeS] clusters would be possible in a nonautocatalytic reaction, we hypothesize a small conformational change in the enzyme, catalyzed by the autocatalyst, which removes a block in the electron flow in either [NiFe] --> [FeS]proximal or the [FeS]proximal --> [FeS]distal reaction step, or removes a block of the penetration of gaseous hydrogen from the surface to the [NiFe] cluster.

Discovery of coding regions in the human genome by integrated proteogenomics analysis workflow.

Proteogenomics enable the discovery of novel peptides (from unannotated genomic protein-coding loci) and single amino acid variant peptides (derived from single-nucleotide polymorphisms and mutations). Increasing the reliability of these identifications is crucial to ensure their usefulness for genome annotation and potential application as neoantigens in cancer immunotherapy. We here present integrated proteogenomics analysis workflow (IPAW), which combines peptide discovery, curation, and validation. IPAW includes the SpectrumAI tool for automated inspection of MS/MS spectra, eliminating false identifications of single-residue substitution peptides. We employ IPAW to analyze two proteomics data sets acquired from A431 cells and five normal human tissues using extended (pH range, 3-10) high-resolution isoelectric focusing (HiRIEF) pre-fractionation and TMT-based peptide quantitation. The IPAW results provide evidence for the translation of pseudogenes, lncRNAs, short ORFs, alternative ORFs, N-terminal extensions, and intronic sequences. Moreover, our quantitative analysis indicates that protein production from certain pseudogenes and lncRNAs is tissue specific.