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18 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 μm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH

Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC–MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as “Mixing QC.” This Mixing QC enables the detection of TMT labeling issues by LC–MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

A Tip-based Workflow for Sensitive IMAC-based Low Nanogram Level Phosphoproteomics

Analyzing the phosphoproteome at nanoscale poses a significant challenge, mainly due to the substantial sample loss from non-specific surface adsorption during the enrichment of low stoichiometric phosphopeptides. Here, we describe a tandem tip-based phosphoproteomics sample preparation method capable of sequential sample cleanup and enrichment without the need for additional sample transfer, thereby minimizing sample loss. Integration of this method to our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches creates a streamlined workflow, enabling sensitive, high-throughput nanoscale phosphoproteomics measurements.

Phosphoproteome, Immobilized metal ion affinity ch↗
DOE OSTI2026

High-throughput Single-Cell Proteomics and Transcriptomics from the Same Cells with a Nanoliter-Scale Spin-Transfer Approach

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

multi 'omics↗
DOE OSTI2023

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We develop a tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3000 (>9500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enables precise quantification of ~600 phosphopeptides from 100 sorted cells (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which in vitro kinase reactions are used to generate targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. More than 7,000 tyrosine phosphorylation sites were quantified from several tens of micrograms of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Abstract Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia are indistinguishable by their cerebrospinal fluid proteomes

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and fibromyalgia have overlapping neurologic symptoms particularly disabling fatigue. This has given rise to the question whether they are distinct central nervous system (CNS) entities or is one an extension of the other. To investigate this, we used unbiased quantitative mass spectrometry-based proteomics to examine the most proximal fluid to the brain, cerebrospinal fluid (CSF). This was to ascertain if the proteome profile of one was the same or different from the other. We examined two separate groups of ME/CFS, one with (n = 15) and one without (n = 15) fibromyalgia. We quantified a total of 2083 proteins using immunoaffinity depletion, tandem mass tag isobaric labelling and offline two-dimensional liquid chromatography coupled to tandem mass spectrometry, including 1789 that were quantified in all the CSF samples. ANOVA analysis did not yield any proteins with an adjusted p value <.05. This supports the notion that ME/CFS and fibromyalgia as currently defined are not distinct entities.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Quantitative proteomics reveals extensive lysine ubiquitination and transcription factor stability states in Arabidopsis

Abstract Protein activity, abundance, and stability can be regulated by post-translational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function; yet, we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich ubiquitinated peptides coupled with isobaric labeling to enable quantification of up to 18-multiplexed samples. This approach identified 17,940 ubiquitinated lysine sites arising from 6,453 proteins from Arabidopsis (Arabidopsis thaliana) primary roots, seedlings, and rosette leaves. Gene ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and metabolism. We determined ubiquitinated lysine residues that directly regulate the stability of three transcription factors, CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) using in vivo degradation assays. Furthermore, codon mutation of CIB1 to create a K166R conversion to prevent ubiquitination, via CRISPR/Cas9-derived adenosine base editing, led to an early flowering phenotype and increased expression of FLOWERING LOCUS T (FT). These comprehensive site-level ubiquitinome profiles provide a wealth of data for future functional studies related to modulation of biological processes mediated by this post-translational modification in plants.

Biochemistry & Molecular Biology↗
DOE OSTI2021

TMTPro Complementary Ion Quantification Increase Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 level

The ability of the TMTPro isobaric labeling reagents to form complementary ions for accurate multiplexed proteomics at the MS2 level was investigated. Human and yeast peptides were labeled in distinct ratios to analyze the effect of interference on the quantification accuracy. A method, TMTProC, was developed and optimized for accurate, sensitive MS2-level quantification of up to 8 conditions in one MS-run.

Stadlmeier, Michael↗
DOE OSTI2021

Resin Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types including cultured cells (e.g. mammalian, prokaryotic) and whole tissues (e.g. heart, lung, muscle) which are initially lysed/homogenized with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol affinity resin which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves our understanding of the role of redox dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

RAC, TMT, thiol redox proteomics, cysteine, PTM st↗
DOE OSTI2023

Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics

There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Here, in this paper, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level

Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Most widely, this issue is alleviated by MS3-based quantification, reducing sensitivity and requiring specialized instrumentation. An alternative approach is quantification by complementary ions, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with the most widely distributed instruments. However, complementary ions of the popular TMT tag form inefficiently and plexing is limited to five channels. In this work, we evaluate and optimize complementary ion quantification for the recently released TMTPro tag, which increases plexing capacity to 8 channels (TMTProC). We find that the beneficial fragmentation properties increase quantification signal five-fold compared to TMT. This increased sensitivity results in ~50% more proteins quantified compared to TMTPro-MS3 and even slightly outperforms TMTPro-MS2. Furthermore, TMTProC quantification is more accurate than TMTPro-MS2 and even superior to TMTPro-MS3. To demonstrate the power of TMTProC, we analyzed a human and yeast interference sample and were able to quantify 13,290 proteins in 24 fractions. Thus, TMTProC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

pmartR 2.0 : A Quality Control, Visualization, and Statistics Pipeline for Multiple Omics Datatypes

The pmartR (https://github.com/pmartR/pmartR) package was designed for the quality control (QC) and analysis of mass spectrometry (MS) data, tailored to specific characteristics of proteomic (isobaric or labelled), metabolomic, and lipidomic datasets. Since its initial release, the tool has been expanded to address the needs of its growing userbase and now includes QC and statistics for nuclear magnetic resonance (NMR) metabolomic data, and leverages the DESeq2, edgeR, and limma-voom R packages for some transcriptomic data analyses. These improvements have made progress towards a unified omics processing pipeline for ease of reporting and streamlined statistical purposes. The package’s statistics and visualization capabilities have also been expanded by adding support for paired data and by integrating pmartR with the trelliscopejs R package for the quick creation of trellis displays (https://github.com/hafen/trelliscopejs). Here, we present relevant examples of each of these enhancements to pmartR and highlight how each new feature benefits the omics community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality

Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One major barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics—currently on the order of 25 mg wet tissue weight, depending on tissue type. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is in excess of 10 million (1E7) peripheral blood mononuclear cells (PBMCs). Throughout the course of a prospective study, this requirement will certainly exceed what is obtainable from many of the individual patients/timepoints. For this reason, we were interested in examining the impact of differential peptide loading across multiplex channels on proteomic data quality. Methods: To achieve this, we tested a range of channel loading amounts (20, 40, 100, 200, and 400 µg of tryptic peptides, or approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification reproducibility and accuracy in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, we found that fewer missing values are observed in TMT channels with higher peptide loading amounts compared to those with lower loading. Moreover, channels with lower loading amounts have greater quantitative variability than channels with higher loading amounts. Statistical analysis of the differences in means among the five loading groups showed that the 20 µg loading group was significantly different from the 400 µg loading group. However, no significant differences were detected among the 40, 100, 200 and 400 µg loading groups. Conclusions: These assessment data demonstrate the practical limits of loading differential quantities of peptides across channels in TMT multiplexes, and provide a basis for designing the optimal clinical proteomics study when specimen quantities are limited.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identifying a novel pathway for extracellular electron uptake in Methanosarcina barkeri using shotgun

The overarching goal of this project was to identify the genetic and/or enzymatic basis for extracellular electron uptake in Methanosarcina barkeri—an organism we previously characterized to be capable of this process using poised potential electrodes and a range of electrochemical techniques. In our first aim, we will utilized proteomic techniques that labels extracellular proteins with free amine groups with biotin and perform a selective purification of these proteins from the rest of the proteome. These were identified using mass spectrometry, providing us insight into the identity of the cell surface proteins, including redox active proteins present during extracellular electron uptake. We will also used an isobaric tagging technique (iTRAQ labeling) of proteomes from different experiment conditions to look at differential proteins expression and better understand the total pathway involved in extracellular electron uptake coupled to methanogenesis. In our final aim, we will investigated the electrochemical nature of electron uptake in co-cultures, compared to our pure culture data sets. We predict the process of electron uptake from electrodes replicates what goes on in nature when these organisms for co-cultures with electrogenic microbes such as Geobacter, and to test this hypothesis we have begun to probe co-cultures electrochemically during direct interspecies electron transfer. Our final aim took much longer than expected due to challenges replicating co-cultures between Geobacter metallireducens and Methanosarcina barkeri. We found the major problem with this work was the inability of the M. barkeri strain we used (and we tried strains from three different labs) to successfully grow on acetate in the absence of hydrogen. As such, we began to use the strain Methanosarcina acetivorans—another close relative that specializes on acetate growth. We are currently in the process of comparing electrochemical observations of this microorganism between pure and co-culture experiments.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2004

A SIFT ion-molecule study of some reactions in Titan's atmosphere. reactions of N(+), N(2)(+), and HCN(+) with CH(4), C(2)H(2), and C(2)H(4)

The results of a study of the ion-molecule reactions of N(+), N(2)(+), and HCN(+) with methane, acetylene, and ethylene are reported. These studies were performed using the FA-SIFT at the University of Canterbury. The reactions studied here are important to understanding the ion chemistry in Titan's atmosphere. N(+) and N(2)(+) are the primary ions formed by photo-ionization and electron impact in Titan's ionosphere and drive Titan's ion chemistry. It is therefore very important to know how these ions react with the principal trace neutral species in Titan's atmosphere: Methane, acetylene, and ethylene. While these reactions have been studied before the product channels have been difficult to define as several potential isobaric products make a definitive answer difficult. Mass overlap causes difficulties in making unambiguous species assignments in these systems. Two discriminators have been used in this study to resolve the mass overlap problem. They are deuterium labeling and also the differences in reactivities of each isobar with various neutral reactants. Several differences have been found from the products in previous work. The HCN(+) ion is important in both Titan's atmosphere and in the laboratory.

Extraterrestrial Environment/chemistry↗