Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.

Developing safe and effective nanoparticles for the delivery of messenger RNA (mRNA) is slow and expensive, partly due to the lack of predictive power of in vitro screening methods and the low-throughput nature of in vivo screening. While DNA barcoding and batch analysis present methods for increasing in vivo screening throughput, they can also result in incomplete or misleading measures of efficacy. Here, we describe a high-throughput and accurate method for the screening of pooled nanoparticle formulations within the same animal. The method uses liquid chromatography with tandem mass spectrometry to detect peptide barcodes translated from mRNAs in nanoparticle-transfected cells. We show the method's applicability by evaluating a library of over 400 nanoparticle formulations with 384 unique ionizable lipids using only nine mice to optimize the formulation of a biodegradable lipid nanoparticle for mRNA delivery to the liver. Barcoding lipid nanoparticles with peptide-encoding mRNAs may facilitate the rapid development of nanoparticles for mRNA delivery to specific cells and tissues.

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavylabeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor -1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/ SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin -like growth factor -1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with dataindependent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.

Protocol for preparing single cells for mass-spec analysis by SCoPE2 as described by Specht et al., doi: 10.1101/665307 (2019) and Specht et al., Genome Biology, doi: 10.1186/s13059-021-02267-5 (2021). For a detailed protocol paper, see Petelski A, Emmott E, Leduc A, Huffman RG, Specht H, Perlman D, Slavov N (2021) Multiplexed single-cell proteomics using SCoPE2 Nature Protocols (in press)

Background Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis. Results To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Conclusions Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.

Abstract INTRODUCTION Pediatric High-Grade Gliomas (PHGG), which include Diffuse Midline Gliomas (DMG), are a leading cause of brain tumor death in children. Our recent work has identified extracellular signal-regulated kinase 5 (ERK5) as a critical mediator of cell survival in DMG, as ERK5 knockdown decreases cell proliferation and extends survival time in orthotopic xenograft mice. Further investigation into the structure of ERK5 shows that it has a kinase domain and, unlike other ERKs, a transactivation domain, and both are important for promoting cell proliferation. HYPOTHESIS AND METHODS We hypothesize that identifying interactors and substrates of ERK5 could identify clinically actionable proteins and provide a more mechanistic insight into ERK5 in the progression of PHGGS. To determine protein–protein associations (PPAs), we employed the proximity-dependent biotin identification (BioID2) method and generated inducible ERK5-BioID2 and ERK2-BioID2 constructs to overcome barriers of conventional screening methods for PPAs. ERK2, similar in structure but much more studied than ERK5, was used as a comparison. Using DIPG lines as proof of principle, we performed streptavidin pull down assays for putative biotinylated ERK PPA and followed with mass spectrometry to identify the ERK2 and ERK5 interactomes. RESULTS Using data-dependent acquisition (DDA), we identified several unique and common interactors of ERK5 compared to ERK2. Through STRING network and pathway analysis, we identified a novel function of ERK5 with respect to membrane trafficking and vesicle transport. Identification of interactors with ERK5 may lead to effective therapeutic combinations. Our current work is focused on validating these interactions and the function of ERK5 in these biological processes. CONCLUSIONS Currently, ERK5 is not as understood as ERK1 or ERK2. Identification of interactors and substrates of ERK5 will further our understanding of PHGG biology, and may lead to identifying druggable targets and pathways.

With the advent of more sensitive and higher duty cycle mass spectrometers, analysis of limited samples, e.g., limited cell populations, microneedle biopsies, extracellular vesicles isolated from minute volumes of physiological fluids (i.e., liquid biopsies) or even single cells, is getting more feasible. However, each step of the analytical workflow, including sample preparation, liquid chromatography (LC) separation, interfacing with mass spectrometry (MS), MS data acquisition, data analysis and data interpretation requires additional advancements to enable deep proteomic profiling of such limited samples. In this study, we investigated MS data acquisition parameters on two different, commonly used mass spectrometers to enhance the sensitivity of proteomic profiling of limited samples. Our experiments indicate that nanoLC-MS/MS-based proteomic analysis of scarce samples requires adjustments of the commonly used default data acquisition settings on a mass spectrometer that work effectively for conventional sample amounts. Here we show that by using specific values of the ion injection time and AGC minimum settings in MS/MS data acquisition depending on the amount of sample results in an increase in the numbers of identified peptides and proteins. We applied commonly used settings, and then altered them to analyze HeLa tryptic digest at the levels of 1, 10 and 100 ng, which are equivalent to approximately 5, 50 and 500 cells on either an Orbitrap QE HFX or a Lumos Fusion (Thermo Fisher Scientific). By changing certain parameters for each sample amount, we were able to identify 2-4-fold more peptides and proteins, and therefore, significantly improve the protein and peptide profiling efficiency, in comparison to the commonly used settings. However, this is only a small step toward ultra-sensitive proteomic profiling. Improvements in LC separation and column material as well as sample preparation are also critical for further advances in highly sensitive analysis.

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that "C-Pro-only" constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.

Recent genome-wide studies have begun to identify gene variants, expression profiles, and regulators associated with neuroticism, anxiety disorders, and depression. We conducted a set of experimental cell culture studies of gene regulation by micro RNAs (miRNAs), based on genome-wide transcriptome, proteome, and miRNA expression data from twenty postmortem samples of lateral amygdala from donors with known neuroticism scores. Using Ingenuity Pathway Analysis and TargetScan, we identified a list of mRNA–protein–miRNA sets whose expression patterns were consistent with miRNA-based translational repression, as a function of trait anxiety. Here, we focused on one gene from that list, which is of particular translational significance in Psychiatry: synaptic vesicle glycoprotein 2A (SV2A) is the binding site of the anticonvulsant drug levetiracetam ((S)-α-Ethyl-2-oxo-1-pyrrolidineacetamide), which has shown promise in anxiety disorder treatments. We confirmed that SV2A is associated with neuroticism or anxiety using an original GWAS of a community cohort ( N = 1,706), and cross-referencing a published GWAS of multiple cohorts (Ns ranging from 340,569 to 390,278). Postmortem amygdala expression profiling implicated three putative regulatory miRNAs to target SV2A : miR-133a, miR-138, and miR-218. Moving from association to experimental causal testing in cell culture, we used a luciferase assay to demonstrate that miR-133a and miR-218, but not miR-138, significantly decreased relative luciferase activity from the SV2A dual-luciferase construct. In human neuroblastoma cells, transfection with miR-133a and miR-218 reduced both endogenous SV2A mRNA and protein levels, confirming miRNA targeting of the SV2A gene. This study illustrates the utility of combining postmortem gene expression data with GWAS to guide experimental cell culture assays examining gene regulatory mechanisms that may contribute to complex human traits. Identifying specific molecular mechanisms of gene regulation may be useful for future clinical applications in anxiety disorders or other forms of psychopathology.

The mission of the ABRF Proteomics Standards Research Group (sPRG) is to identify and implement technical standards that reflect the ABRF's commitment to accuracy, clarity, and consistency in the field of proteomics. There is broad interest in quantifying protein phosphorylation alterations in cellular signaling pathways, but their transient nature and low abundance makes their analysis challenging. Here we follow up on our sPRG heavy-labeled phosphopeptide standard study designed to address issues encountered in phosphopeptide experiments. This standard is constructed of 150 heavy-labeled phosphopeptides spanning seven different signaling pathways and will be useful to test the effectiveness of phosphopeptide enrichment workflows, as an internal instrument and chromatography calibrant, and as a pre-built biological assay for a wide variety of signaling pathways. In our initial characterization and validation of this standard (sPRG 2018-2019 study), we mixed the standard into an activated HeLa tryptic digest and distributed the mixture to participants with optimized and standardized enrichment and acquisition methods. Data independent acquisition (DIA) was performed on 8 gas phase fractionated injections. As an extension of this initial characterization and to prepare for a larger biological study, we present progress towards a universal single inject targeted data acquisition method using our standard which aims to be accurate, sensitive, scalable, and easily implemented and executed without the need for retention time scheduling. Two approaches were taken that both target the heavy isotope labeled internal standard peptides and trigger a mass offset scan of the endogenous light. The first method, QuanDirect, monitors for heavy y1 fragments and can be implemented on tribrid instrument platforms. The second method, SureQuant, monitors for select fragment ions and can be implemented on both tribrid and hybrid instruments.

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release ofmolecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.

The fate and physiology of individual cells are controlled by protein interactions. We recently developed SCoPE-MS, a method for direct analysis of proteomes of single cells by LC-MS/MS. SCoPE-MS is enabled by isobaric-tag multiplexing of peptides from single cells together with carrier material, which serves both to minimize sample losses to equipment surfaces and enhance peptide identifications. Yet, our ability to quantitatively analyze proteins in single cells has remained limited. To overcome this barrier, we developed SCoPE2. SCoPE2 lowers cost and hands-on time by introducing automated and miniaturized sample preparation while increasing quantitative accuracy using only commercially available equipment and reagents. Additionally, SCoPE2 accomplishes increased sample preparation throughput and increased measurement throughput. Using SCoPE2, we quantified over 2,700 proteins in 1,018 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allowed us to discern single cells by cell type. Parallel measurements of transcripts by 10x Genomics scRNA-seq indicate that most genes had similar responses at the protein and RNA levels, though the responses of hundreds of genes differed. In this talk, we present users with how to begin adopting SCoPE2 in their lab. Executing a SCoPE2 experiment has similarities to conventional proteomics sample preparation as well as pitfalls and deviations unique to working with single cells. We discuss successful strategies, including study design and reagent selection, how to avoid common pitfalls, and the range of common equipment that can be used to execute SCoPE2 sample prep. Additionally, we discuss approaches to optimizing LC-MS/MS instrumentation for SCoPE2 samples. We hope to give users ideas about how to successfully design and execute a SCoPE2 experiment to facilitate the broad adoption of automated and quantitative single-cell analysis of proteins by mass-spectrome.

ABSTRACT During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called UPR (Unfolded Protein Response) that functions to maintain ER (endoplasmic reticulum) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification (PTM) called Fic-mediated Adenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an AMP (adenosine monophosphate) to the ER chaperone, BiP, to alter the cell’s UPR-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE - alpha-Synuclein (αSyn), a presynaptic protein involved in Parkinson’s disease (PD). Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in PD models. We show that HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation in vitro , suggesting a possible mechanism by which cells cope with αSyn toxicity. HIGHLIGHTS Aggregated forms of the presynaptic protein αSyn cause neurotoxicity and induce ER stress in cellular and animal models of Parkinson’s disease. We have identified αSyn as a novel target for the human Fic protein, HYPE, a key regulator of ER homeostasis. HYPE adenylylates αSyn and reduces the aggregation of recombinant αSyn Fic-mediated adenylylation/AMPylation is a possible mechanism by which cells cope with αSyn toxicity. Graphic Abstract

The mission of the ABRF Proteomics Standards Research Group (sPRG) is to identify and implement technical standards that reflect the ABRF's commitment to accuracy, clarity, and consistency in the field of proteomics. There is broad interest in quantifying protein phosphorylation alterations in cellular signaling pathways under different conditions. The transient nature and low abundance of many phosphorylation sites makes this analysis challenging. Here we report on the follow up of the two-year sPRG study designed to target various issues encountered in phosphopeptide experiments. We have constructed a pool of heavy-labeled phosphopeptides that will enable core facilities to rapidly develop assays. Our pool contains over 150 phosphopeptides that have been previously observed in mass spectrometry data sets. The specific peptides have been chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling pathways: AMPK, death and apoptosis, ErbB, insulin/IGF-1, mTOR, PI3K/AKT, and stress (p38/SAPK/JNK). We feel this pool will enable researchers to test the effectiveness of their enrichment workflows and to provide a benchmark for a cross lab study. This standard should be helpful in number of ways, including providing a complete workflow solution for phosphopeptide enrichment, as an internal enrichment and chromatography calibrant, and as a pre-built biological assay for a wide variety of signaling pathways. Previously, we mixed the standard into an activated HeLa tryptic digest and distributed the mixture to over 60 ABRF member and nonmember laboratories around the world. We asked participants to enrich phosphopeptides out of the HeLa background and report ratios of the heavy phosphopeptides to the endogenous levels. In the current study, we continue validation of the standard within various RG group/ABRF members' laboratories. The aim of this follow up study is to provide reagents, an optimized phosphopeptide enrichment protocol, instrument acquisition method parameters, and data analysis templates.

During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic-mediatedadenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE—alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation invitro, suggesting a possible mechanism by which cells cope with αSyn toxicity.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides a unique in situ chemical profile that can include drugs, nucleic acids, metabolites, lipids, and proteins. MSI of individual cells (of a known cell type) affords a unique insight into normal and disease-related processes and is a prerequisite for combining the results of MSI and other single-cell modalities (e.g. mass cytometry and next-generation sequencing). Technological barriers have prevented the high-throughput assignment of MSI spectra from solid tissue preparations to their cell type. These barriers include obtaining a suitable cell-identifying image (e.g. immunohistochemistry) and obtaining sufficiently accurate registration of the cell-identifying and MALDI-MS images. This study introduces a technique that overcame these barriers by assigning cell type directly from mass spectra. We hypothesized that, in MSI from mice with a defined fluorescent protein expression pattern, the fluorescent protein's molecular ion could be used to identify cell cohorts. A method was developed for the purification of enhanced yellow fluorescent protein (EYFP) from mice. To determine EYFP's molecular mass for MSI studies, we performed intact mass analysis and characterized the protein's primary structure and post-translational modifications through various techniques. MALDI-MSI methods were developed to enhance the detection of EYFP in situ, and by extraction of EYFP's molecular ion from MALDI-MS images, automated, whole-image assignment of cell cohorts was achieved. This method was validated using a well-characterized mouse line that expresses EYFP in motor and sensory neurons and should be applicable to hundreds of commercially available mice (and other animal) strains comprising a multitude of cell-specific fluorescent labels.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

After a radiological incident, there is an urgent need for fast and reliable bioassays to identify radiation-exposed individuals within the first week post exposure. This study aimed to identify candidate radiation-responsive protein biomarkers in human lymphocytes in vivo using humanized NOD scid gamma (Hu-NSG) mouse model. Three days after X-irradiation (0–2 Gy, 88 cGy/min), human CD45+ lymphocytes were collected from the Hu-NSG mouse spleen and quantitative changes in the proteome of the human lymphocytes were analysed by mass spectrometry. Forty-six proteins were differentially expressed in response to radiation exposure. FDXR, BAX, DDB2 and ACTN1 proteins were shown to have dose-dependent response with a fold change greater than 2. When these proteins were used to estimate radiation dose by linear regression, the combination of FDXR, ACTN1 and DDB2 showed the lowest mean absolute errors (≤0.13 Gy) and highest coefficients of determination (R2 = 0.96). Biomarker validation studies were performed in human lymphocytes 3 days after irradiation in vivo and in vitro. In conclusion, this is the first study to identify radiation-induced human protein signatures in vivo using the humanized mouse model and develop a protein panel which could be used for the rapid assessment of absorbed dose 3 days after radiation exposure.

Proteomics characterization of biofluids, such as urine and plasma, has been explored for the discovery of predictive, prognostic, and mechanistic biomarkers of diseases and tissue injury. Here we describe comprehensive characterization of protein cargos from cell-derived secreted vesicles (extracellular vesicles or exosome) for biomarker discovery using the mass spectrometry-based technology.