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PubMed2025

Proviral functions of HMGB1 in HAdV-C5 replication compartments.

UNLABELLED: Human adenoviruses (HAdVs) induce significant reorganization of the nuclear environment, leading to the formation of virus-induced subnuclear structures known as replication compartments (RCs). Within these RCs, viral genome replication, gene expression, and modulation of cellular antiviral responses are tightly coordinated, making them valuable models for studying virus-host interactions. In a recent study, we analyzed the protein composition of HAdV type 5 (HAdV-C5) RCs isolated from infected primary cells at different time points during infection using quantitative proteomics. We identified several chromatin modifiers, including the high-mobility group box 1 protein (HMGB1) as components associated with RCs and demonstrated that HMGB1 can be relocalized to RCs from different HAdV species, thereby modulating viral replication in a species-specific manner. In the present work, using click-chemistry and proximity ligation assays, we discovered that HMGB1 localizes to sites of DNA replication within RCs and that its interaction with DBP in RCs is dependent on both DNA replication and RC assembly. HMGB1-knockdown experiments demonstrated that HMGB1 is required for efficient viral gene expression. However, despite its proviral role in viral replication, we found that HMGB1 levels decreased in late stages of infection due to transcriptional downregulation. Furthermore, by overexpressing HMGB1, we showed that this regulation of HMGB1 levels during infection is critical for optimal HAdV-C5 replication. These results highlight the complex regulatory relationship between HMGB1 and HAdV-C5 infection. IMPORTANCE: In an extensive proteomics analysis, we found that HMGB1, an important cellular chromatin protein, was enriched in adenovirus replication compartments. In this study, we aimed to better understand the role of HMGB1 in the infection process of a human DNA virus, HAdV-C5. We tested different virus types, including some with specific gene deletions and mutations. Our results showed that during infection, HMGB1 levels decreased because the virus suppressed its production. Despite this, even at lower levels, HMGB1 still helped the virus replicate by interacting with key viral proteins and DNA at sites where the virus is actively replicating. Overall, our findings highlight how HMGB1 plays a crucial role in facilitating efficient virus replication, making it an important factor in the infection process.

HMGB1 Protein↗
PubMed2026

Mocravimod as a repurposing drug against clinical isolates of Staphylococcus aureus by targeting cell membrane.

UNLABELLED: Staphylococcus aureus infections, particularly those caused by multidrug-resistant strains and associated with biofilm formation, pose a major therapeutic challenge in clinical practice. The objective of this study was to evaluate the antibacterial and antibiofilm activity of mocravimod (KRP-203), an FDA-approved S1P receptor modulator, against clinical S. aureus isolates and to explore its underlying mechanism of action. The antibacterial activity of KRP-203 was assessed against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) using MIC determination, time-kill assays, and biofilm inhibition models. KRP-203 exhibited strong bactericidal activity against planktonic MSSA and MRSA, with MIC values ranging 6.25-50μM. Time-kill assays demonstrated rapid bacterial eradication at 8× MIC within 2 h, showing superior killing kinetics compared with vancomycin. At sub-inhibitory concentrations, KRP-203 inhibited biofilm formation by up to 70% and reduced viable bacterial counts in mature biofilms by >2.5 logs. To elucidate the antibacterial mechanism, whole-genome sequencing and quantitative proteomic analyses were performed. These analyses revealed mutations in membrane-associated genes, including glnQ and BCAT, and significant alterations in proteins related to membrane integrity and redox regulation. Consistently, functional assays confirmed that KRP-203 disrupts bacterial cell membrane, as evidenced by dose-dependent membrane depolarization, increased permeability, and direct binding to cardiolipin and phosphatidylglycerol. Molecular docking further predicted a favorable interaction between KRP-203 and GlnQ. In conclusion, KRP-203 demonstrated notable antibacterial and antibiofilm activity against S. aureus, likely through membrane integrity disruption. While these findings highlight its potential as a repurposed antibacterial agent, further studies are required to fully elucidate its molecular targets, optimize antibacterial efficacy, and evaluate its in vivo safety profile. IMPORTANCE: Antibiotic resistance and the formation of biofilms, which protect bacteria from medications and immunological responses, present the significant challenges for the clinical treatment of Staphylococcus aureus infections. This study reveals mocravimod hydrochloride (KRP-203), a clinically approved drug initially intended to treat leukemia, as a viable new candidate against S. aureus infection. KRP-203 quickly kills both drug-susceptible and resistant S. aureus, including difficult-to-treat biofilm-associated cells. Its membrane-disrupting activity quickly kills drug-resistant bacteria while also destroying biofilm formations, presenting a dual action rarely accomplished by conventional antibiotics. Critically, KRP-203's established safety profile in human studies may hasten its repurposing as a new weapon against biofilm-associated infections, providing possible solutions for chronic and drug-resistant S. aureus infections where existing treatments commonly fail.

Biofilms↗
PubMed2006

In vivo measurement of synthesis rate of multiple plasma proteins in humans.

Advances in quantitative proteomics have facilitated the measurement of large-scale protein quantification, which represents net changes in protein synthesis and breakdown. However, measuring the rate of protein synthesis is the only way to determine the translational rate of gene transcripts. Here, we report a technique to measure the rate of incorporation of amino acids from ingested protein labeled with stable isotope into individual plasma proteins. This approach involves three steps: 1) production of stable isotope-labeled milk whey protein, oral administration of this intrinsically labeled protein, and subsequent collection of blood samples; 2) fractionation of the plasma and separation of the individual plasma proteins by a combination of anion exchange high-pressure liquid chromatography and gel electrophoresis; and 3) identification of individual plasma proteins by tandem mass spectrometry and measurement of stable isotopic enrichment of these proteins by gas chromatography-mass spectrometry. This method allowed the measurement of the fractional synthesis rate (FSR) of 29 different plasma proteins by using the same precursor pool. We noted a 30-fold difference in FSR of different plasma proteins with a wide range of physiological functions. This approach offers a tremendous opportunity to study the regulation of plasma proteins in humans in many physiological and pathological states.

Adult↗
PubMed2026

The Charcot-Marie-Tooth Neuropathy (CMTX3) Complex Structural Variation Causes Differential SOX3 Spatiotemporal Expression.

Charcot-Marie-Tooth (CMT) neuropathy is a clinically and genetically heterogeneous group of diseases characterized by the length-dependent axonal degeneration of peripheral nerves. We previously mapped a rare form of X-linked CMT, CMTX3, to a 5.7-Mb interval on chromosome Xq26.3-q27.1 and excluded the coding region of all known genes in the linkage interval for mutations. Whole genome sequencing subsequently identified a 78-kb region of chromosome 8q24.3 that had been duplicated and inserted into the CMTX3 locus between the genes HAPSTR2 and SOX3. The 78-kb insertion, which contains a partial transcript of ARHGAP39, fully segregated in families with CMTX3 and was absent in neurologically normal controls. To retain the CMTX3 insertion and investigate its consequences in appropriate neuronal tissue, we generated induced pluripotent stem cells (iPSCs) from CMTX3 fibroblasts. Using bulk RNA sequencing of patient-derived spinal motor neurons, ARHGAP39 was deemed nonpathogenic by excluding both the formation of novel fusion transcripts and dosage effects from the partial duplication. Subsequent NanoString expression analyses of candidate genes within the CMTX3 locus, across different stages of neuronal differentiation, identified spatiotemporal dysregulation of SOX3. NanoString showed reduced SOX3 expression in patient iPSCs. RNA sequencing detected SOX3 downregulation in CMTX3 neuroepithelial progenitor cells, which was further confirmed by quantitative proteomics. Given the early onset and relatively rapid progression of CMTX3, these data prioritise SOX3 as a leading candidate gene, consistent with its role as one of the earliest transcription factors expressed in the developing nervous system and a key regulator of neuronal fate.

Humans↗
PubMed2005

Novel mass spectrometric methods for evaluation of plasma angiotensin converting enzyme 1 and renin activity.

This article demonstrates the applicability of quantitative proteomics to assays of proteolytic enzyme activity. A novel assay was developed for measurement of renin and angiotensin-converting enzyme (ACE) activity in plasma. The method was validated in animal models associated with alterations of the renin angiotensin system (RAS). Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with a ProteinChip Array technology, plasma renin and ACE1 could be measured in <0.5 microL of plasma. Plasma is incubated with peptide substrates for renin and ACE, tetradecapeptide (TDP), and angiotensin I (Ang I), respectively. The reactions mixtures are spotted onto the ProteinChip WCX2 and detected using SELDI-TOF-MS. Peak height or area under curve for TDP, Ang I, and angiotensin II (Ang II) peaks are measured. There was a linear relationship between disappearance of substrate and appearance of products for both renin and ACE (R2=0.95 to 0.98). ACE1 activity was blocked with chelating agents (EDTA and 1,10 phenanthrolene), indicating action of a metalloprotease. The ACE1 inhibitor, captopril, selectively blocked ACE1. Renin activity was specifically blocked with renin inhibitor and was not affected by phenanthrolene or captopril. Animal models tested were Ang AT1a receptor-deficient and streptozotocin (STZ) diabetic mice. Plasma renin activity was increased >2-fold in AT1a(-/-) as compared with AT1a(+/+). In STZ diabetic mice, ACE1 was increased 2-fold as compared with controls. The advantage of the method is that it is tagless, does not require additional purification steps, and is extremely sensitive. The approach can be multiplexed and used for identification of novel substrates/inhibitors of the RAS.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

Optimisation of the two-dimensional gel electrophoresis protocol using the Taguchi approach.

BACKGROUND: Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE) for separation and characterisation of complex protein mixtures. Among the difficulties associated with this approach is the solubilisation of protein mixtures for isoelectric focusing (IEF). To find the optimal formulation of the multi-component IEF rehydration buffer (RB) we applied the Taguchi method, a widely used approach for the robust optimisation of complex industrial processes, to determine optimal concentrations for the detergents, carrier ampholytes and reducing agents in RB for 2DE using commercially supplied immobilised pH gradient (IPG) gel strips. RESULTS: Our optimisation resulted in increased protein solubility, improved resolution and reproducibility of 2D gels, using a wide variety of samples. With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved. Moreover, with improved sample loading and resolution, analysis of individual spots by immunoblotting and mass spectrometry revealed previously uncharacterised posttranscriptional modifications in a variety of chromatin proteins. CONCLUSIONS: While the optimised RB (oRB) is specific to the gels and analysis approach we use, our use of the Taguchi method should be generally applicable to a broad range of electrophoresis and analysis systems.

Journal Article↗
PubMed2006

Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites.

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of 15N-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized 15N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic 15N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.

Journal Article↗
PubMed2005

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1.

Addition of exogenous ceramide causes a significant displacement of cholesterol in lipid raft model membranes. However, whether ceramide-induced cholesterol displacement is sufficient to alter the protein composition of caveolin-enriched lipid raft membranes is unknown. Therefore, we examined whether increasing endogenous ceramide levels with bacterial sphingomyelinase (bSMase) depleted cholesterol and changed the protein composition of caveolin-enriched membranes (CEMs) isolated from immortalized Schwann cells. bSMase increased ceramide levels severalfold and decreased the cholesterol content of detergent-insoluble CEMs by 25-50% within 2 h. To examine the effect of ceramide on the protein composition of the CEMs, we performed a quantitative proteomic analysis using stable isotope labeling of cells in culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although ceramide rapidly depleted lipid raft cholesterol, the levels of the cholesterol binding protein caveolin-1 (Cav-1) decreased by 25% only after 8 h. Importantly, replenishing the cells with cholesterol rapidly reversed the loss of Cav-1 from the CEMs. Ceramide-induced cholesterol depletion increased the association of 5'-nucleotidase and ATP synthase beta-subunit with the CEMs but had a minimal effect on changing the abundance of other lipid raft proteins, such as flotillin-1 and G-proteins. These results suggest that the ceramide-induced loss of cholesterol from CEMs may contribute to altering the lipid raft proteome.

5'-Nucleotidase↗
PubMed2025

Tumor Signatures of Physical Fitness: Insights from a Preclinical Model.

PURPOSE: Cardiorespiratory fitness (CRF) and muscle strength are associated with cancer risk/mortality in adults. However, there is yet no evidence for pediatric tumors. This study investigated the association of CRF and muscle strength with several tumor-related phenotypes in an aggressive childhood malignancy, high-risk neuroblastoma. METHODS: Twelve mice-bearing orthotopic high-risk neuroblastomas were studied. CRF and muscle strength were assessed using treadmill and grip strength testing, respectively. The following tumor-related outcomes were studied: survival, clinical severity, tumor weight/volume, metastasis, and intratumor immune infiltrates. In addition, tumor samples underwent quantitative proteomic analysis via liquid chromatography-tandem mass spectrometry. Spearman correlations (or logistic regression) were performed between CRF/muscle strength and the abovementioned variables. Proteins that were significantly correlated with CRF or muscle strength were mapped into protein-protein interaction (PPI) networks using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. RESULTS: CRF was inversely correlated with clinical severity score ( r = -0.657, P = 0.020). Of 6840 identified tumor proteins, 76 correlated significantly with CRF (19 positively, 57 negatively), whereas 194 correlated with muscle strength (97 positively, 97 negatively). Proteins correlated with CRF were primarily involved in metabolic and structural pathways, including angiotensinogen and elastin. In turn, muscle strength-associated proteins were more abundant and included keratin family proteins (e.g., keratin, type I cytoskeletal 14, and type II cytoskeletal 5), proteins involved in cell adhesion (e.g., desmoglein-1-alpha), and translational regulators (e.g., eukaryotic initiation factor 4A). Network analysis revealed significant enrichment in structural organization and cellular adhesion pathways. CONCLUSIONS: Besides the association of CRF with clinical severity of the tumor, distinct novel tumor proteomic signatures associated with CRF and muscle strength were identified, highlighting potential mechanisms linking physical fitness with childhood cancer biology.

Muscle Strength↗
PubMed2026

A Bayesian framework for multivariate differential analysis.

Differential analysis is a routine procedure in the statistical analysis toolbox across many applied fields, including quantitative proteomics, the main illustration of the present paper. The state-of-the-art limma approach uses a hierarchical formulation with moderated-variance estimators for each analyte directly injected into the t-statistic. While standard hypothesis testing strategies are recognised for their low computational cost, allowing for quick extraction of the most differential among thousands of elements, they generally overlook key aspects such as handling missing values, inter-element correlations, and uncertainty quantification. The present paper proposes a fully Bayesian framework for differential analysis, leveraging a conjugate hierarchical formulation for both the mean and the variance. Inference is performed by computing the posterior distribution of compared experimental conditions and sampling from the distribution of differences. This approach provides well-calibrated uncertainty quantification at a similar computational cost as hypothesis testing by leveraging closed-form equations. Furthermore, a natural extension enables multivariate differential analysis that accounts for possible inter-element correlations. We also demonstrate that, in this Bayesian treatment, missing at random data should generally be ignored in univariate settings, and further derive a tailored approximation that handles multiple imputation for the multivariate setting. We argue that probabilistic statements in terms of effect size and associated uncertainty are better suited to practical decision-making. Therefore, we finally propose simple and intuitive inference criteria, such as the overlap coefficient, which express group similarity as a probability rather than traditional, and often misleading, p-values. The performance of this approach is evaluated through an extensive empirical study using both synthetic and controlled real-world proteomics datasets. Overall, we believe that this Bayesian framework for (multivariate) differential analysis provides a valuable and intuitive counterpart to standard methods at a comparable computational cost.

Bayes Theorem↗
PubMed2026

Prolonged In Vitro Expansion Shapes the Neuro-Supportive Potential of Jaw Periosteum Secretomes: Implications for Secretome Product Quality.

Nerve injuries are frequent complications of complex oral and maxillofacial surgical procedures, particularly following extensive tumor resections. Secretome-based, cell-free therapies derived from mesenchymal stromal cells have emerged as promising regenerative approaches; however, robust manufacturing requires the identification of critical quality attributes (CQAs) that ensure product potency and consistency. The influence of replicative senescence during in vitro expansion on the quality of jaw periosteum-derived mesenchymal stromal cell (JPC) secretomes has not yet been established. This study investigated whether the expansion state of JPCs affects the composition and neuro-supportive potency of their secretomes. Secretomes from four independent JPC donors were collected separately at early and late passages, pooled within each passage-specific preparation, and applied to human induced pluripotent stem cell-derived neurons. Neuronal survival, neurite outgrowth, and neuronal marker expression were assessed as functional readouts. Secretome composition was characterized by quantitative proteomics and enzyme-linked immunosorbent assay (ELISA) of selected senescence-associated secretory phenotype (SASP) factors. Secretomes derived from early-passage JPCs significantly enhanced neuronal survival and neurite outgrowth, whereas late-passage secretomes displayed reduced neuro-supportive activity. Proteomic profiling identified a pronounced shift toward inflammatory and stress-associated signaling, whereas performed ELISAs confirmed senescence-associated remodeling of the secretome, including increased abundance of SASP-associated factors in late-passage preparations. These findings demonstrate that prolonged in vitro expansion profoundly influences both the composition and biological potency of JPC-derived secretomes. Collectively, this study identifies the passage-associated senescence-like phenotype of JPCs as a key determinant of secretome quality and supports its consideration as a critical quality attribute for the manufacturing and standardization of JPC-derived secretome products. Monitoring and controlling the expansion state of JPCs may therefore be essential to ensure the consistency, potency, and clinical translation of secretome-based regenerative therapies.

Humans↗
PubMed2026

Chromatin Remodeling Subunit ARID1A Negatively Regulates the Malignant Progression of Gastrointestinal Stromal Tumors by Targeting the MEMO1 Promoter.

Gastrointestinal stromal tumors (GISTs) are the most common sarcomas of the alimentary tract and are primarily characterized by malignant progression, a major cause of mortality. AT-rich interaction domain 1A (ARID1A), a core component of the chromatin-remodeling SWI/SNF complex, has been found to correlate with GIST tumor grade, although the underlying mechanism remains unclear. Its frequent inactivation across diverse cancer types reveals pleiotropic roles that intersect multiple hallmarks of cancer. In this study, we aimed to investigate the potential relationship between ARID1A and malignant progression in GISTs, as well as the underlying mechanism. Western blotting, real-time polymerase chain reaction, and immunohistochemistry were used to assess ARID1A expression in GIST tissues. Cell Counting Kit-8 (CCK-8) assays were performed to evaluate cell proliferation. Wound-healing and Transwell assays were conducted to assess cell migration and invasion. Flow cytometry was used to analyze apoptosis and cell cycle distribution. Label-free quantitative proteomics and chromatin immunoprecipitation sequencing (ChIP-seq) were employed to identify top candidate downstream targets of ARID1A. ARID1A expression was decreased in high-risk GIST tissues. Furthermore, ARID1A knockdown in GIST cells promoted proliferation and metastasis both in vitro and in vivo, and led to reduced apoptosis and impaired cell cycle arrest. We further demonstrated that ARID1A suppresses GIST proliferation and metastasis by inhibiting MEMO1 expression and inactivating the ERK1/2 signaling pathway. Notably, this regulatory axis was observed in KIT-null GIST cells, indicating that the ARID1A-MEMO1 pathway may function independently of canonical KIT signaling. Thus, ARID1A inhibits malignant progression in GISTs, providing new insights into its role in the prevention and treatment of human GISTs and suggesting its potential as a biomarker of malignant progression in GISTs.

Humans↗
PubMed2026

Small extracellular vesicles reflect senescence progression in human bone marrow-derived mesenchymal stem cells during hollow fiber bioreactor culture.

Prolonged three-dimensional culture exposes stem cells to sustain microenvironmental and mechanical stresses that can promote aging- and senescence-associated phenotypic alterations. This study examined how long-term expansion of human bone marrow-derived mesenchymal stem cells (BMSCs) in a hollow fiber bioreactor (HFB) influences cellular senescence and the molecular composition of secreted small extracellular vesicles (sEVs). During extended HFB culture, BMSCs exhibited progressive morphological flattening and cytoskeletal disorganization, accompanied by increased senescence-associated &#x3b2;-galactosidase activity and immunophenotypic remodeling characterized by reduced fluorescence intensity and spatial redistribution of canonical MSC markers, consistent with a stress-adapted, early senescence-associated cellular state. In parallel, sEVs were collected longitudinally over 40 days and characterized by nanoparticle tracking analysis, immunoblotting, and quantitative proteomics. While vesicle size, marker expression, and yield remained stable throughout culture, proteomic profiling revealed pronounced, phase-dependent remodeling of sEV cargo, including coordinated alterations in oxidative stress-related processes, lysosomal and extracellular matrix-associated pathways, and relative depletion of cytoskeletal and translational components. Notably, these vesicular signatures closely mirrored senescence-associated changes observed at the cellular level. The strong correspondence between cellular phenotypes and sEV proteomic profiles establishes vesicle analysis as a convergent and noninvasive readout of BMSC aging, enabling sensitive monitoring of senescence progression while reducing reliance on parallel, labor-intensive cellular assays. Collectively, these findings indicate that prolonged HFB culture promotes a controlled, stress-associated senescence program in BMSCs and position sEV proteomic profiling as a robust approach for assessing stem cell aging dynamics during long-term three-dimensional bioreactor culture.

Mesenchymal Stem Cells↗
PubMed2005

[Development of the new tumor markers].

Serum tumor markers are invasive diagnostic tools for malignant tumors and have been commonly used for purposes of screening, prognosis and selection of treatments. In order to develop a new marker, gene expression analysis technologies such as DNA microarray, differential display, cDNA subtraction, and serial analysis of gene expression, which enable investigators to obtain comprehensive data with respect to gene-expression profiles, are progressing rapidly. Several studies have already demonstrated the usefulness of these techniques for identifying novel cancer-related genes and for classifying human cancers at the molecular level. However, significant differences between the abundance ratio of the mRNA transcript and the corresponding protein product are observed for many genes. Also, the protein level in serum is affected by many physiological conditions and related circumstances, not only by mRNA levels in the cell. Therefore, quantitative proteomics technology based on high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis is being developed. One of our goals is the serological evaluation for the new candidate proteins derived from the information of the above transcriptome and/or proteome analysis as a tumor marker. In this report, I will discuss the practical process of the investigation with special regards to a new protein named ALCAN which has been studied through cDNA cloning, preparation of recombinant protein and monoclonal antibodies, construction of immune assay system, and clinical evaluation.

Biomarkers, Tumor↗
PubMed2002

Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry.

A primary challenge in proteome measurements is to be able to detect, identify, and quantify the extremely complex mixtures of proteins. The relative abundances of interest span at least six orders of magnitude for mammalian proteomes, and this constitutes an intractable challenge for high throughput proteome studies. We have recently described a new approach, Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), which is based upon the selective ejection of the most abundant species to expand the dynamic range of Fourier transform ion cyclotron resonanace (FTICR) measurements. The basis of our approach is on-the-fly data-dependent selective ejection of highly abundant species, followed by prolonged accumulation of remaining low-abundance species in a quadrupole external to the FTICR ion trap. Here we report the initial implementation of this approach with high efficiency capillary reverse phase LC separations and high magnetic field electrospray ionization FTICR mass spectrometry for obtaining enhanced coverage in quantitative measurements for mammalian proteomes. We describe the analysis of a sample derived from a tryptic digest of proteins from mouse B16 cells cultured in both natural isotopic abundance and 15N-labeled media. The FTICR mass spectrometric analysis allows the assignment of peptide pairs (corresponding to the two distinctive versions of each peptide), and thus provides the basis for quantiative measurements when one of the two proteomes in the mixture is perturbed or altered in some fashion. We show that implementation of the DREAMS approach allows assignment of approximately 80% more peptide pairs, thus providing quantitative information for approximately 18,000 peptide pairs in a single analysis.

Animals↗
PubMed2006

A review of quantitative methods for proteomic studies.

An overview is provided of six strategies for relative or absolute quantitation of protein abundances that are widely used in proteomic studies. Strengths and limitations are discussed. Four of these involve stable isotope labeling and isotope ratio measurements by mass spectrometry. In another, mass spectra are used to deconvolute overlapping peptide HPLC peaks to provide relative quantitation based on peak areas. The sixth provides relative abundances of proteins based on 2-D gel arrays. It should be noted that these strategies measure peptide and protein abundances, and cannot directly assess changes in regulation or expression.

Amino Acid Sequence↗
PubMed2000

Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins.

Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and PGDS (prostaglandin D2 synthase) were the other major compounds secreted. The most abundant proteins found in the luminal fluid were albumin and the secreted proteins: lactoferrin, PGDS, GPX, HE1/CTP, and hexosaminidase. Three main secretory epididymal regions were identified from the protein pattern, i.e., regions E0-E2, E3-E5, and E6-E9. Region E0-E2 was characterized by the secretion of clusterin (53%), PGDS (44%), and GPX (6%). Region E3-E5 had the highest number of secreted proteins, the highest protein concentrations (60-80 mg/ml), and the highest spermatocrit value (85%). Lactoferrin (60% in E4), clusterin (29% in E3), hexosaminidase (10% in E3), and procathepsin D (6.9% in E4) were the most abundant proteins in this region. Region E6-E9, in which few region-specific secreted compounds were found, was characterized by a high quantity of lactoferrin in the luminal fluid (2-14 mg/ml). Comparison between the secretion of the major proteins and their concentrations in the lumen throughout the organ showed that the behavior of each protein is specific, in particular for the three isoforms of clusterin.

Amino Acid Sequence↗
PubMed2003

A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture.

Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells. It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail. However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample. We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels. In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation. We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.

Amino Acid Sequence↗