Monitoring Both Extended and Tryptic Forms of Stable Isotope-Labeled Standard Peptides Provides an Internal Quality Control of Proteolytic Digestion in Targeted Mass Spectrometry-Based Assays.

Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.

A multiplexed assay for quantifying immunomodulatory proteins supports correlative studies in immunotherapy clinical trials.

Introduction: Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens. Methods: Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins. Results and discussion: The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community.

Internal Standard Triggered-Parallel Reaction Monitoring Mass Spectrometry Enables Multiplexed Quantification of Candidate Biomarkers in Plasma.

Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Previously, mass spectrometry-based approaches have been used to fill this gap but feature poor quantitative performance and were generally limited to hundreds of proteins. Here, we demonstrate the capability of an internal standard triggered-parallel reaction monitoring (IS-PRM) assay to greatly expand the numbers of candidates that can be tested with improved quantitative performance. The assay couples immunodepletion and fractionation with IS-PRM and was developed and implemented in human plasma to quantify 5176 peptides representing 1314 breast cancer biomarker candidates. Characterization of the IS-PRM assay demonstrated the precision (median % CV of 7.7%), linearity (median R2 > 0.999 over 4 orders of magnitude), and sensitivity (median LLOQ < 1 fmol, approximately) to enable rank-ordering of candidate biomarkers for validation studies. Using three plasma pools from breast cancer patients and three control pools, 893 proteins were quantified, of which 162 candidate biomarkers were verified in at least one of the cancer pools and 22 were verified in all three cancer pools. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

Prochlorococcus extracellular vesicles: molecular composition and adsorption to diverse microbes.

Extracellular vesicles are small (~50-200 nm diameter) membrane-bound structures released by cells from all domains of life. While vesicles are abundant in the oceans, their functions, both for cells themselves and the emergent ecosystem, remain a mystery. To better characterize these particles - a prerequisite for determining function - we analysed the lipid, protein, and metabolite content of vesicles produced by the marine cyanobacterium Prochlorococcus. We show that Prochlorococcus exports a diverse array of cellular compounds into the surrounding seawater enclosed within discrete vesicles. Vesicles produced by two different strains contain some materials in common, but also display numerous strain-specific differences, reflecting functional complexity within vesicle populations. The vesicles contain active enzymes, indicating that they can mediate extracellular biogeochemical reactions in the ocean. We further demonstrate that vesicles from Prochlorococcus and other bacteria associate with diverse microbes including the most abundant marine bacterium, Pelagibacter. Together, our data point toward hypotheses concerning the functional roles of vesicles in marine ecosystems including, but not limited to, possibly mediating energy and nutrient transfers, catalysing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species.

Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

Morphological Plasticity in a Sulfur-Oxidizing Marine Bacterium from the SUP05 Clade Enhances Dark Carbon Fixation.

Sulfur-oxidizing bacteria from the SUP05 clade are abundant in anoxic and oxygenated marine waters that appear to lack reduced sources of sulfur for cell growth. This raises questions about how these chemosynthetic bacteria survive across oxygen and sulfur gradients and how their mode of survival impacts the environment. Here, we use growth experiments, proteomics, and cryo-electron tomography to show that a SUP05 isolate, "Candidatus Thioglobus autotrophicus," is amorphous in shape and several times larger and stores considerably more intracellular sulfur when it respires oxygen. We also show that these cells can use diverse sources of reduced organic and inorganic sulfur at submicromolar concentrations. Enhanced cell size, carbon content, and metabolic activity of the aerobic phenotype are likely facilitated by a stabilizing surface-layer (S-layer) and an uncharacterized form of FtsZ-less cell division that supports morphological plasticity. The additional sulfur storage provides an energy source that allows cells to continue metabolic activity when exogenous sulfur sources are not available. This metabolic flexibility leads to the production of more organic carbon in the ocean than is estimated based solely on their anaerobic phenotype.IMPORTANCE Identifying shifts in microbial metabolism across redox gradients will improve efforts to model marine oxygen minimum zone (OMZ) ecosystems. Here, we show that aerobic morphology and metabolism increase cell size, sulfur storage capacity, and carbon fixation rates in "Ca Thioglobus autotrophicus," a chemosynthetic bacterium from the SUP05 clade that crosses oxic-anoxic boundaries.

Heterotrophic carbon metabolism and energy acquisition in Candidatus Thioglobus singularis strain PS1, a member of the SUP05 clade of marine Gammaproteobacteria.

A hallmark of the SUP05 clade of marine Gammaproteobacteria is the ability to use energy obtained from reduced inorganic sulfur to fuel autotrophic fixation of carbon using RuBisCo. However, some SUP05 also have the genetic potential for heterotrophic growth, raising questions about the roles of SUP05 in the marine carbon cycle. We used genomic reconstructions, physiological growth experiments and proteomics to characterize central carbon and energy metabolism in Candidatus Thioglobus singularis strain PS1, a representative from the SUP05 clade that has the genetic potential for autotrophy and heterotrophy. Here, we show that the addition of individual organic compounds and 0.2 µm filtered diatom lysate significantly enhanced the growth of this bacterium. This positive growth response to organic substrates, combined with expression of a complete TCA cycle, heterotrophic pathways for carbon assimilation, and methylotrophic pathways for energy conversion demonstrate strain PS1's capacity for heterotrophic growth. Further, our inability to verify the expression of RuBisCO suggests that carbon fixation was not critical for growth. These results highlight the metabolic diversity of the SUP05 clade that harbours both primary producers and consumers of organic carbon in the oceans and expand our understanding of specific pathways of organic matter oxidation by the heterotrophic SUP05.

Stress response of a marine ammonia-oxidizing archaeon informs physiological status of environmental populations.

High representation by ammonia-oxidizing archaea (AOA) in marine systems is consistent with their high affinity for ammonia, efficient carbon fixation, and copper (Cu)-centric respiratory system. However, little is known about their response to nutrient stress. We therefore used global transcriptional and proteomic analyses to characterize the response of a model AOA, Nitrosopumilus maritimus SCM1, to ammonia starvation, Cu limitation and Cu excess. Most predicted protein-coding genes were transcribed in exponentially growing cells, and of ~74% detected in the proteome, ~6% were modified by N-terminal acetylation. The general response to ammonia starvation and Cu stress was downregulation of genes for energy generation and biosynthesis. Cells rapidly depleted transcripts for the A and B subunits of ammonia monooxygenase (AMO) in response to ammonia starvation, yet retained relatively high levels of transcripts for the C subunit. Thus, similar to ammonia-oxidizing bacteria, selective retention of amoC transcripts during starvation appears important for subsequent recovery, and also suggests that AMO subunit transcript ratios could be used to assess the physiological status of marine populations. Unexpectedly, cobalamin biosynthesis was upregulated in response to both ammonia starvation and Cu stress, indicating the importance of this cofactor in retaining functional integrity during times of stress.

Photochemical and Nonphotochemical Transformations of Cysteine with Dissolved Organic Matter.

Cysteine (Cys) plays numerous key roles in the biogeochemistry of natural waters. Despite its importance, a full assessment of Cys abiotic transformation kinetics, products and pathways under environmental conditions has not been conducted. This study is a mechanistic evaluation of the photochemical and nonphotochemical (dark) transformations of Cys in solutions containing chromophoric dissolved organic matter (CDOM). The results show that Cys underwent abiotic transformations under both dark and irradiated conditions. Under dark conditions, the transformation rates of Cys were moderate and were highly pH- and temperature-dependent. Under UVA or natural sunlight irradiations, Cys transformation rates were enhanced by up to two orders of magnitude compared to rates under dark conditions. Product analysis indicated cystine and cysteine sulfinic acid were the major photooxidation products. In addition, this study provides an assessment of the contributions of singlet oxygen, hydroxyl radical, hydrogen peroxide, and triplet dissolved organic matter to the CDOM-sensitized photochemical oxidation of Cys. The results suggest that another unknown pathway was dominant in the CDOM-sensitized photodegradation of Cys, which will require further study to identify.

Photooxidation of the Antimicrobial, Nonribosomal Peptide Bacitracin A by Singlet Oxygen under Environmentally Relevant Conditions.

Bacitracin is a mixture of nonribosomal peptides (NRPs) that is extensively used as an antibiotic in both human and veterinary medicine. Despite its widespread use over the past six decades, very few studies have addressed the environmental fate of bacitracin and zinc-bacitracin complexes. In this study, the photochemical transformation of bacitracin components (i.e., cyclic dodecapeptides) in the aquatic environment was investigated. A high resolution mass spectrometry (HRMS)-based approach enabled monitoring of the photochemical degradation kinetics of individual bacitracin components, investigation of the relative contribution of reactive oxygen species (e.g., singlet oxygen, (1)O2) in dissolved organic matter-sensitized photoreactions, and identification of oxidative modifications in bacitracin photoproducts. The results of this study support the hypothesis that indirect photochemical oxidation of the histidine (His) residue by (1)O2 is a major degradation pathway for bacitracin A, the most potent congener of the mixture. Furthermore, the photooxidation rate of bacitracin A with (1)O2 decreased upon bacitracin A coordination with Zn(2+), demonstrating that the photochemistry of metal-bound His is different from that of metal-free His. Overall, these results provide insight into the fate of bacitracin components in the aquatic environment and highlight the potential of utilizing this HRMS-based methodology to study transformations of other environmentally relevant NRPs.

Assessing the Indirect Photochemical Transformation of Dissolved Combined Amino Acids through the Use of Systematically Designed Histidine-Containing Oligopeptides.

Photooxidation is an important abiotic transformation pathway for amino acids (AAs) in sunlit waters. Although dissolved free AAs are well studied, the photooxidation of dissolved combined AAs (DCAAs) remains poorly investigated. This study is a systematic investigation of the effect of neighboring photostable AA residues (i.e., aliphatic, cationic, anionic, or aromatic residues) on the environmental indirect photochemical transformation of histidine (His) in His-containing oligopeptides. The pKa values of His residues in the studied oligopeptides were found to be between 4.3 and 8.1. Accordingly, the phototransformation rate constants of the His-containing oligopeptides were highly pH-dependent in an environmentally relevant pH range with higher reactivity for neutral His than for the protonated species. The photostable AA residues significantly modulated the photoreactivity of oligopeptides either through altering the accessibility of His to photochemically produced oxidants or through shifting the pKa values of His residues. In addition, the influence of neighboring photostable AA residues on the sorption-enhanced phototransformation of oligopeptides in solutions containing chromophoric dissolved organic matter (CDOM) was assessed. The constituent photostable AA residues promoted sorption of His-containing oligopeptides to CDOM macromolecules through electrostatic attraction, hydrophobic effects, and/or low-barrier hydrogen bonds, and subsequently increased the apparent phototransformation rate constants by up to 2 orders of magnitude.

Enhanced Indirect Photochemical Transformation of Histidine and Histamine through Association with Chromophoric Dissolved Organic Matter.

Photochemical transformations greatly affect the stability and fate of amino acids (AAs) in sunlit aquatic ecosystems. Whereas the direct phototransformation of dissolved AAs is well investigated, their indirect photolysis in the presence of chromophoric dissolved organic matter (CDOM) is poorly understood. In aquatic systems, CDOM may act both as sorbent for AAs and as photosensitizer, creating microenvironments with high concentrations of photochemically produced reactive intermediates, such as singlet oxygen (1O2). This study provides a systematic investigation of the indirect photochemical transformation of histidine (His) and histamine by 1O2 in solutions containing CDOM as a function of solution pH. Both His and histamine showed pH-dependent enhanced phototransformation in the CDOM systems as compared to systems in which model, low-molecular-weight 1O2 sensitizers were used. Enhanced reactivity resulted from sorption of His and histamine to CDOM and thus exposure to elevated 1O2 concentrations in the CDOM microenvironment. The extent of reactivity enhancement depended on solution pH via its effects on the protonation state of His, histamine, and CDOM. Sorption-enhanced reactivity was independently supported by depressed rate enhancements in the presence of a cosorbate that competitively displaced His and histamine from CDOM. Incorporating sorption and photochemical transformation processes into a reaction rate prediction model improved the description of the abiotic photochemical transformation rates of His in the presence of CDOM.

Environmental Photochemistry of Amino Acids, Peptides and Proteins.

Amino acids, peptides and proteins are central building blocks of life and of key importance in the biogeochemistry of aquatic ecosystems. In sunlit surface waters, amino acid-based molecules at different levels of structural organization are susceptible to transformation by both direct photochemical reactions and indirect processes caused by photochemically produced reactive oxygen species (e.g. hydroxyl radical or singlet oxygen). Photochemical transformation processes can thereby affect the availability of these crucial nutrient sources in aquatic ecosystems, inhibit the function of microbial extracellular enzymes, or even promote the degradation of amino acid-based pollutant molecules. In this article, the environmental photochemistry of amino acids, peptides and proteins in aquatic systems is reviewed.

Reactivity differences of combined and free amino acids: quantifying the relationship between three-dimensional protein structure and singlet oxygen reaction rates.

It has long been appreciated that the photooxidation kinetics of amino acid (AA) residues in an intact protein differ from those of free AAs due to differences in the local steric microenvironment, such as its location in the three-dimensional structure. Yet there are only a few studies that have quantified the effect of protein structure on the photochemical reactivity of its residues. This is important for predicting phototransformation rates of AAs in aquatic environments where AAs in combined forms (e.g., oligopeptides and proteins) are more abundant than free AAs. In this work, the photochemical reactivity differences between free and combined AAs were assessed. Singlet oxygen ((1)O2) reaction kinetics of individual photooxidizable residues in the protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The results suggest that the (1)O2 accessibility of residues in intact GAPDH has a profound effect on their photodegradation kinetics and for histidine residues can explain most of the variation in (1)O2 reactivity. Additionally, (1)O2-accessibile surface area values of residues calculated from protein crystal structure data are useful in predicting their reaction rates in GAPDH. This work illustrates a new approach to assess the differential photochemical reactivity of AA-based biomolecules in natural environments or engineered applications.

Direct photochemistry of three fluoroquinolone antibacterials: norfloxacin, ofloxacin, and enrofloxacin.

Fluoroquinolone (FQ) antibacterial compounds are frequently detected in the aquatic environment, and photodegradation is expected to play an important role in FQ fate in some sunlit surface waters. This study investigated the direct aquatic photochemistry of three FQs: norfloxacin, ofloxacin, and enrofloxacin. The direct photolysis rate of each drug exhibited strong pH dependence when exposed to simulated sunlight. For each FQ, direct photolysis rates and total light absorbance were used to calculate quantum yields for each of three environmentally relevant protonation states: a cationic, a zwitterionic, and an anionic form. In each case, quantum yields of the species varied significantly. The quantum yield for the zwitterionic form was 2-3 times higher than that of the anionic form and over an order of magnitude higher than that of the cationic form. Antibacterial activity assays were used to determine whether the loss of parent FQ due to photolysis led to loss of activity. Norfloxacin and ofloxacin photoproducts were found to be inactive, whereas enrofloxacin photoproducts were found to retain significant activity. These results are important for aiding in predictions of the potential impacts of FQs in surface waters.

Environmental photochemistry of tylosin: efficient, reversible photoisomerization to a less-active isomer, followed by photolysis.

The environmental photochemical kinetics of tylosin, a common veterinary macrolide antibiotic and growth promoter, were investigated under simulated sunlight. An efficient, reversible photoisomerization was characterized using kinetic, mass spectrometry, and proton nuclear magnetic resonance data. The photoisomerization was confirmed to occur by a rotation about the distal alkene of the ketodiene functionality. Concurrent forward (quantum yield = 0.39 +/- 0.09) and back (quantum yield = 0.32 +/- 0.08) reactions lead to a photochemical equilibrium near a tylosin/photoisomer ratio of 50:50, completed in less than 2 min under a spectrum equivalent to noontime, summer sunlight. The activity of the isomer for the inhibition of Escherichia coli DH5alpha growth was observed to be less than that of tylosin. On a longer time scale than that of isomerization, the isomer mixture undergoes photolysis with a quantum yield of (1.4 +/- 0.3) x 10(-3). The observed quantum yields and UV-vis absorbance data allow for the prediction of the photochemical behavior of tylosin in most environmental systems. Indirect photosensitization was not a significant loss process in solutions of Suwannee River fulvic acid with concentrations from 1 to 20 mg L(-1).