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CMMB-based isopropanol gradient peptide fractionation (CIF) enables rapid and robust off-line peptide mixture fractionation in bottom-up proteomics

ABSTRACT: HEK293 cells lysates fractionated by various different methods

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: James A. Wohlschlegel

PROVIDER: MSV000085458 | MassIVE | Fri May 22 10:35:00 BST 2020

REPOSITORIES: MassIVE

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Publications

Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics.

Deng Weixian W Sha Jihui J Plath Kathrin K Wohlschlegel James A JA

Molecular & cellular proteomics : MCP 20210119

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of coeluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias toward high-abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based ...[more]

PMID: 33476790

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Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics.

Project description:Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of coeluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias toward high-abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or carboxylate-modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange and reversed phase-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH reversed phase. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low-abundance bait proteins.

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Preparative capillary electrophoresis (CE) fractionation of protein digests improves protein and peptide identification in bottom-up proteomics.

Project description:Reversed-phase liquid chromatography (RPLC) is widely used to reduce sample complexity prior to mass spectrometry (MS) analysis in bottom-up proteomics. Improving peptide separation in complex samples enables lower-abundance proteins to be identified. Multidimensional separations that combine orthogonal separation modes improve protein and peptide identifications over RPLC alone. Here we report a preparative capillary electrophoresis (CE) fractionation method that combines CE and RPLC separations. Using this method, we demonstrate improved protein and peptide identification in a tryptic digest of E. coli cell lysate, with 132 ± 33% more protein identifications and 185 ± 65% more peptide identifications over non-fractionated samples. Fractionation enables detection of lower-abundance proteins in this complex sample. We demonstrate improved coverage of ovarian cancer biomarker MUC16 isolated from conditioned cell media, with 6.73% sequence coverage using CE fractionation compared to 2.74% coverage without preparative fractionation. This new method will allow researchers performing bottom-up proteomics to harness the advantages of CE separations while using widely available LC-MS/MS instrumentation.

| S-EPMC9210495 | biostudies-literature

Bottom-up formation of robust gold carbide.

Project description:A new phenomenon of structural reorganization is discovered and characterized for a gold-carbon system by in-situ atomic-resolution imaging at temperatures up to 1300 K. Here, a graphene sheet serves in three ways, as a quasi transparent substrate for aberration-corrected high-resolution transmission electron microscopy, as an in-situ heater, and as carbon supplier. The sheet has been decorated with gold nanoislands beforehand. During electron irradiation at 80 kV and at elevated temperatures, the accumulation of gold atoms has been observed on defective graphene sites or edges as well as at the facets of gold nanocrystals. Both resulted in clustering, forming unusual crystalline structures. Their lattice parameters and surface termination differ significantly from standard gold nanocrystals. The experimental data, supported by electron energy loss spectroscopy and density-functional theory calculations, suggests that isolated gold and carbon atoms form - under conditions of heat and electron irradiation - a novel type of compound crystal, Au-C in zincblende structure. The novel material is metastable, but surprisingly robust, even under annealing condition.

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Bottom-Up Design Approach for OBOC Peptide Libraries.

Project description:One-bead-one-compound peptide libraries, developed following the top-down experimental approach, have attracted great interest in the identification of potential ligands or active peptides. By exploiting a reverse experimental design approach based on the bottom-up strategy, we aimed to develop simplified, maximally diverse peptide libraries that resulted in the successful characterization of mixture components. We show that libraries of 32 and 48 components can be successfully detected in a single run using chromatography coupled to mass spectrometry (UPLC-MS). The proposed libraries were further theoretically evaluated in terms of their composition and physico-chemical properties. By combining the knowledge obtained on single libraries we can cover larger sequence spaces and provide a controlled exploration of the peptide chemical space both theoretically and experimentally. Designing libraries by using the bottom-up approach opens up the possibility of rationally fine-tuning the library complexity based on the available analytical methods.

| S-EPMC7435479 | biostudies-literature

Gradient microfluidics enables rapid bacterial growth inhibition testing.

Project description:Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask).

| S-EPMC3988682 | biostudies-literature

Rapid sample preparation for bottom-up proteome

Project description:a rapid sample preparation method has petential for daily clinic diagnosis

2020-09-15 | PXD021476 |

Learning beyond-pairwise interactions enables the bottom-up prediction of microbial community structure.

Project description:Understanding the assembly of multispecies microbial communities represents a significant challenge in ecology and has wide applications in agriculture, wastewater treatment, and human healthcare domains. Traditionally, studies on the microbial community assembly focused on analyzing pairwise relationships among species; however, neglecting higher-order interactions, i.e., the change of pairwise relationships in the community context, may lead to substantial deviation from reality. Herein, we have proposed a simple framework that incorporates higher-order interactions into a bottom-up prediction of the microbial community assembly and examined its accuracy using a seven-member synthetic bacterial community on a host plant, duckweed. Although the synthetic community exhibited emergent properties that cannot be predicted from pairwise coculturing results, our results demonstrated that incorporating information from three-member combinations allows the acceptable prediction of the community structure and actual interaction forces within it. This reflects that the occurrence of higher-order effects follows consistent patterns, which can be predicted even from trio combinations, the smallest unit of higher-order interactions. These results highlight the possibility of predicting, explaining, and understanding the microbial community structure from the bottom-up by learning interspecies interactions from simple beyond-pairwise combinations.

| S-EPMC10873592 | biostudies-literature

Rapid preparation of human blood plasma for bottom-up proteomics analysis.

Project description:This protocol offers step-by-step instructions for preparation of raw blood plasma for liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis in clinical proteomics studies. The technique is simple, robust, and reproducible, and the entire transformation from plasma proteins to desalted tryptic peptides takes only 3-4 h. The protocol ensures efficient denaturation of native proteases that, in combination with the speediness of the procedure, prevents non-specific and irreproducible cleavage of digested peptides. The protocol can be adopted for large-scale studies and automation. For complete details on the use and execution of this protocol, please refer to Overmyer et al. (2020).

| S-EPMC8503661 | biostudies-literature

Two-dimensional combinatorial screening enables the bottom-up design of a microRNA-10b inhibitor.

Project description:The RNA motifs that bind guanidinylated kanamycin A (G Kan A) and guanidinylated neomycin B (G Neo B) were identified via two-dimensional combinatorial screening (2DCS). The results of these studies enabled the "bottom-up" design of a small molecule inhibitor of oncogenic microRNA-10b.

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Trimodal chromatography enables facile peptide fractionation

Project description:We evaluated the utility of a trimodal stationary phase (Trinity P1) in offline peptide fractionation, and found it performed similarly to ion exchange or high pH reversed phase based techniques. It is orthogonal to low pH reversed phase and uses MS compatible mobile phase solvents.

2017-08-07 | PXD006188 | Pride

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