Ultrahigh resolution mass spectrometry, such as Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS), can resolve thousands of molecular ions in complex organic matrices. A ...Compound Identification Algorithm (CIA) was previously developed for automated elemental formula assignment for natural organic matter (NOM). In this work, we describe software Formularity with a user-friendly interface for CIA function and newly developed search function Isotopic Pattern Algorithm (IPA). While CIA assigns elemental formulas for compounds containing C, H, O, N, S, and P, IPA is capable of assigning formulas for compounds containing other elements. We used halogenated organic compounds (HOC), a chemical class that is ubiquitous in nature as well as anthropogenic systems, as an example to demonstrate the capability of Formularity with IPA. A HOC standard mix was used to evaluate the identification confidence of IPA. Tap water and HOC spike in Suwannee River NOM were used to assess HOC identification in complex environmental samples. Strategies for reconciliation of CIA and IPA assignments were discussed. Software and sample databases with documentation are freely available.
Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due ...to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand.
SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity ...assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.
SUMMARY
The establishment of the nitrogen‐fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we ...utilized laser ablation electrospray ionization‐mass spectrometry (LAESI‐MS) for in situ metabolic profiling of wild‐type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl‐acyl carrier protein desaturase (sacpd‐c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+) and ineffective (nifH mutant, fix−) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd‐c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI‐MS for high‐throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.
Significance Statement
Biological nitrogen fixation is important for sustainable agriculture. The present study examined metabolites in soybean nodules using laser ablation electrospray ionization‐mass spectrometry, a high‐throughput and powerful approach for in situ metabolic profiling. The results provide information regarding those metabolic pathways affected in nodules disrupted in the symbiosis as a result of mutation.
Characterization of the metabolic heterogeneity in cell populations requires the analysis of single cells. Most current methods in single-cell analysis rely on cell manipulation, potentially altering ...the abundance of metabolites in individual cells. A small sample volume and the chemical diversity of metabolites are additional challenges in single-cell metabolomics. Here, we describe the combination of fiber-based laser ablation electrospray ionization (f-LAESI) with 21 T Fourier transform ion cyclotron resonance mass spectrometry (21TFTICR-MS) for in situ single-cell metabolic profiling in plant tissue. Single plant cells infected by bacteria were selected and sampled directly from the tissue without cell manipulation through mid-infrared ablation with a fine optical fiber tip for ionization by f-LAESI. Ultrahigh performance 21T-FTICR-MS enabled the simultaneous capture of isotopic fine structures (IFSs) for 47 known and 11 unknown compounds, thus elucidating their elemental compositions from single cells and providing information on metabolic heterogeneity in the cell population.
The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response ...to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca
-imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism.
Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development ...of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)–based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano–liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50–100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.
Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. ...The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown. We propose here an ambitious initiative to define the human proteome, that is, to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented along with a call for improved technologies in a two-pronged strategy to the Human Proteoform Project.
•Column length was found as an important factor for top-down proteomic RPLC separation.•Long (≥1m) columns can provide peak capacities of >400 for resolving proteoforms.•Both porous and superficially ...porous particles were effective to separate proteins. Particles with 200–450Å pores enabled chromatographing >100kDa proteoforms.•C1-C18-bonded phases had their own limits for eluting various sizes of proteoforms.
Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. Herein, we report use of long (≥1M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5MPa or 14Kpsi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5–5μm particles were used as packings and long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50kDa. Larger proteoforms (50–110kDa) were chromatographed on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200–400Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ∼900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC–MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Our initial data indicate that MS detection and fragmentation inefficiencies provided by current high-resolution mass spectrometers are key challenges for characterization of larger proteoforms.
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