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PubMed2006

Systematically assessing the influence of 3-dimensional structural context on the molecular evolution of mammalian proteomes.

The 3-dimensional (3D) structural context of amino acid residues in a protein could significantly impact the level of selective constraint on the residues. Here, by analyzing 767 mammalian proteins, we systematically investigate how various 3D structural contexts influence selective constraint. The structural contexts we examined include solvent accessibility, secondary structure, and intramolecular residue-residue interactions. Through this analysis, we offer quantitative information on how 3D structural contexts affect the level of selective constraint.

Amino Acids↗

PubMed2004

Use of mass spectrometry to identify protein biomarkers of disease severity in the synovial fluid and serum of patients with rheumatoid arthritis.

OBJECTIVE: To identify a panel of candidate protein biomarkers of rheumatoid arthritis (RA) that can predict which patients will develop erosive, disabling disease. METHODS: A 2-step proteomic approach was used for biomarker discovery and verification. In the first step, 2-dimensional liquid chromatography-coupled tandem mass spectrometry was used to generate protein profiles of synovial fluid (SF) from patients with either erosive RA (n = 5) or nonerosive RA (n = 5). In the second step, the selected candidate markers were verified using quantitative multiple reaction monitoring mass spectrometry in sera of patients with erosive RA (n = 15) or nonerosive RA (n = 15) and of healthy controls (n = 15). RESULTS: Through differential profiling of proteins in the <40-kd portion of the SF proteome, we selected 33 prospective candidate biomarkers from a total of 418 identified proteins. Among the proteins that were elevated in the SF of patients with erosive RA were C-reactive protein (CRP) and 6 members of the S100 protein family of calcium-binding proteins. Significantly, levels of CRP, S100A8 (calgranulin A), S100A9 (calgranulin B), and S100A12 (calgranulin C) proteins were also elevated in the serum of patients with erosive disease compared with patients with nonerosive RA or healthy individuals. CONCLUSION: Several potential protein marker candidates have been identified for prognosis of the erosive form of RA. This study demonstrates the facility of using protein mass spectrometry in SF and serum for global discovery and verification of clinically relevant sets of disease biomarkers.

Adult↗

PubMed2026

Dynamic lysine acetylation and succinylation of platelet proteins regulates platelet storage lesion: mechanistic insights from multi-omics.

OBJECTIVES: Platelet storage lesion (PSL) severely impairs platelet function during storage, presenting a major hurdle in transfusion medicine; however, the dynamic interplay between global proteomic changes and post-translational modifications (PTMs) underlying these functional deteriorations remains insufficiently characterized. Here, we report the first comprehensive multi-omics analysis integrating global proteomics, acetylomics, and succinylomics to dissect the molecular dynamics during platelet storage. METHODS: We performed quantification of global proteomics, acetylome and succinylome based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification. Dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively. RESULTS: We systematically characterized 3,609 proteins, 1,308 acetylation sites, and 1,947 succinylation sites across multiple storage time points (D1, D3, D5, D7). We distinct temporal patterns of post-translational modifications, with succinylation showing more extensive coverage than acetylation in platelets. Pathway enrichment analysis revealed extensive metabolic reprogramming involving complement activation, energy metabolism, and cellular detoxification processes. The identification of specific motif patterns provided mechanistic insights into the functional specificity of these modifications. Random forest machine learning identified 20 core regulatory proteins representing critical nodes in PSL development. Furthermore, we employed real - time quantitative polymerase chain reaction (RT - QPCR) to measure the expression levels of key genes related to platelet function and PTM - associated pathways. CONCLUSION: By mapping the interplay between proteomic abundance shifts and PTM dynamics, this study provides a multidimensional understanding of PSL, establishing a foundational framework for optimizing storage protocols and enhancing transfusion safety.

Blood Platelets↗

PubMed2006

Modern proteomes contain putative imprints of ancient shifts in trace metal geochemistry.

Because of the rise in atmospheric oxygen 2.3 billion years ago (Gya) and the subsequent changes in oceanic redox state over the last 2.3-1 Gya, trace metal bioavailability in marine environments has changed dramatically. Although theorized to have influenced the biological usage of metals leaving discernable genomic signals, a thorough and quantitative test of this hypothesis has been lacking. Using structural bioinformatics and whole-genome sequences, the Fe-, Zn-, Mn-, and Co-binding metallomes of 23 Archaea, 233 Bacteria, and 57 Eukarya were constructed. These metallomes reveal that the overall abundances of these metal-binding structures scale to proteome size as power laws with a unique set of slopes for each Superkingdom of Life. The differences in the power describing the abundances of Fe-, Mn-, Zn-, and Co-binding proteins in the proteomes of Prokaryotes and Eukaryotes are similar to the theorized changes in the abundances of these metals after the oxygenation of oceanic deep waters. This phenomenon suggests that Prokarya and Eukarya evolved in anoxic and oxic environments, respectively, a hypothesis further supported by structures and functions of Fe-binding proteins in each Superkingdom. Also observed is a proliferation in the diversity of Zn-binding protein structures involved in protein-DNA and protein-protein interactions within Eukarya, an event unlikely to occur in either an anoxic or euxinic environment where Zn concentrations would be vanishingly low. We hypothesize that these conserved trends are proteomic imprints of changes in trace metal bioavailability in the ancient ocean that highlight a major evolutionary shift in biological trace metal usage.

Animals↗

PubMed2001

Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE*) is a new development in protein detection for two-dimensional gels. Using mouse liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP)-treated), we have determined the quantitative variation in the 2-D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster 2D-Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently observed protein changes resulting from APAP-treatment were identified by mass spectrometry. Several of these can be rationalised based on available data on the mechanism of APAP hepatotoxicity but others cannot, indicating that proteomics can provide further insights into the biochemical basis of APAP toxicity.

Acetaminophen↗

PubMed2005

Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography.

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

Antineoplastic Agents↗

PubMedDate not supplied

Mature dendritic cells secrete exosomes with strong ability to induce antigen-specific effector immune responses.

Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes which transfer functional MHC-peptide complexes to other DCs. Since immature and mature DCs induce different functional T cell responses (i.e., tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that immature and mature murine DCs secrete morphologically similar exosomes. Extensive proteomic analysis of the two exosome populations showed identical overall protein composition, and provided an exhaustive image of the protein composition of DC-derived exosomes. By quantitative analysis, however, exosomes from mature DCs proved enriched in MHC class II, B7.2, ICAM-1, and depleted in MFG-E8, as compared to immature exosomes. In functional T cell stimulation assays, exosomes secreted by mature DCs were 50- to 100-fold more potent than exosomes from immature DCs, both in vitro and in vivo. MHC class II and ICAM-1 were necessary for the increased immune activity of exosomes secreted by mature DCs. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.

Animals↗

PubMed2006

Characterization of the human pancreatic islet proteome by two-dimensional LC/MS/MS.

The pancreatic beta-cell plays a central role in the maintenance of glucose homeostasis and in the pathogenesis of both type 1 and type 2 diabetes mellitus. Elucidation of the insulin secretory defects observed in diabetes first requires a better understanding of the complex mechanisms regulating insulin secretion, which are only partly understood. While there have been reports detailing proteomic analyses of islet cell lines or isolated rodent islets, the information gained is not always applicable to humans. Therefore, definition of the human islet proteome could contribute to a better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the confident identification of 29,021 different tryptic peptides covering 3365 proteins (> or =2 unique peptide identifications per protein). As expected, the three major islet hormones (insulin, glucagon, and somatostatin) were detected, as well as various beta-cell enriched secretory products, ion channels, and transcription factors. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was observed, including the integrin signaling cascade and the MAP kinase, NF-kappa beta, and JAK/STAT pathways. The resulting peptide reference library provides a resource for future higher throughput and quantitative studies of islet biology.

Chromatography, Liquid↗

PubMed2006

Proteomic analysis of tumor establishment and growth in the B16-F10 mouse melanoma model.

The B16-F10 mouse model of melanoma is a widely used model to study many aspects of cancer biology and therapeutics in a solid tumor. Melanomas aggressively progress within a dynamic microenvironment containing in addition to tumor cells, stroma cells and components such as fibroblasts, immune cells, vascular cells, extracellular matrix (ECM) and extracellular molecules. The goal of this study was to elucidate the processes of tumor progression by identifying differentially expressed proteins in the tumor mass during specific stages of tumor growth. A comparative proteome analysis was performed on B16-F10 derived tumors in C57BL/6 mice at days 3, 5, 7, and 10. Statistical approaches were used to determine quantitative differential protein expression at each tumor time stage. Hierarchical clustering of 44 protein spots (p < 0.01) revealed a progressive change in the tumor mass when all 4 time stages were classified together, but there was a clear switch in expression of these proteins between the day 5 and the day 7 tumors. A trend analysis showed 53 protein spots (p < 0.001) following 6 predominant kinetic paths of expression as the tumor progressed. The protein spots were then identified using MALDI-TOF mass spectrometry. Proteins involved in glycolysis, inflammation, wounding, superoxide metabolism, and chemotaxis increased during tumorigenesis. From day 3 to day 7 VEGF and active cathepsin D were induced 7-fold and 4-fold, respectively. Proteins involved in electron transport, protein folding, blood coagulation, and transport decreased during tumorigenesis. This work illustrates changes in the biology of the B16-F10 tumor mass during tumor progression.

Animals↗

PubMed2006

Preliminary approaches for the development of a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method for the detection and label-free semi-quantitation of the main storage proteins of Lupinus albus in foods.

Since recent literature has indicated that white lupin (Lupinus albus) may be a useful source of hypocholesterolemic proteins to be used in functional food formulation, our final goal is the development of a fast and automated high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the detection and the label-free semi-quantitation of the main lupin globulins in lupin foods and food ingredients. We present here some preliminary results in this direction. As a first step a total protein extract (TPE-WF) from lupin flakes was pre-fractionated by anion-exchange chromatography and each fraction was digested with trypsin and analyzed by HPLC/ESI-MS/MS. Subsequently, the tryptic digest of TPE-WF was directly analyzed by HPLC/ESI-MS/MS without any pre-fractionation. Eventually, in order to test the applicability of the method to real samples, a lupin beverage and two lupin protein isolates were analyzed. Both Mascot and Spectrum Mill MS Proteomics Workbench software were used to identify the protein composition in these samples and Spectrum Mill was used also to test the possibility of developing a label-free semi-quantitation method based on peptide hits. Encouraging results were obtained especially in the detection of the hypocholesterolemic component beta-conglutin.

Amino Acid Sequence↗

PubMed2006

Identification of diagnostic markers for tuberculosis by proteomic fingerprinting of serum.

BACKGROUND: We investigated the potential of proteomic fingerprinting with mass spectrometric serum profiling, coupled with pattern recognition methods, to identify biomarkers that could improve diagnosis of tuberculosis. METHODS: We obtained serum proteomic profiles from patients with active tuberculosis and controls by surface-enhanced laser desorption ionisation time of flight mass spectrometry. A supervised machine-learning approach based on the support vector machine (SVM) was used to obtain a classifier that distinguished between the groups in two independent test sets. We used k-fold cross validation and random sampling of the SVM classifier to assess the classifier further. Relevant mass peaks were selected by correlational analysis and assessed with SVM. We tested the diagnostic potential of candidate biomarkers, identified by peptide mass fingerprinting, by conventional immunoassays and SVM classifiers trained on these data. FINDINGS: Our SVM classifier discriminated the proteomic profile of patients with active tuberculosis from that of controls with overlapping clinical features. Diagnostic accuracy was 94% (sensitivity 93.5%, specificity 94.9%) for patients with tuberculosis and was unaffected by HIV status. A classifier trained on the 20 most informative peaks achieved diagnostic accuracy of 90%. From these peaks, two peptides (serum amyloid A protein and transthyretin) were identified and quantitated by immunoassay. Because these peptides reflect inflammatory states, we also quantitated neopterin and C reactive protein. Application of an SVM classifier using combinations of these values gave diagnostic accuracies of up to 84% for tuberculosis. Validation on a second, prospectively collected testing set gave similar accuracies using the whole proteomic signature and the 20 selected peaks. Using combinations of the four biomarkers, we achieved diagnostic accuracies of up to 78%. INTERPRETATION: The potential biomarkers for tuberculosis that we identified through proteomic fingerprinting and pattern recognition have a plausible biological connection with the disease and could be used to develop new diagnostic tests.

Adolescent↗

PubMed2005

Quantitative mass spectrometric identification of isomers applying coherent laser control.

Mass spectrometry (MS) is one of the oldest and most trusted analytical methods for chemical identification. Advances in biology, such as metabolic analysis and proteomics, have fueled a growing number of refinements in this method. Unfortunately, isomers, for example, o- and p-xylene, are seldom identifiable by MS because they produce identical spectra. Time-consuming and less sensitive multidimensional methods are subsequently required for structural determination. The sensitivity of MS coupled with shaped femtosecond laser pulses that control molecular fragmentation and ionization results in a new, fast, and reproducible method for molecular identification which is used here to distinguish positional and geometric isomer compounds and quantify their relative concentration in mixtures.

Journal Article↗

PubMed2006

Proteomic approaches to bacterial differentiation.

Mass spectrometry-based proteomics has been used extensively to explore the proteomes of various organisms, and this technology is now being applied to the characterization of bacterial species. Predominantly, two methods emerge as leaders in this application. Intact protein profiling creates fingerprints of bacterial species which can be used for differentiation and tracking over time. Peptide-centric approaches, analyzed after enzymatic digestion, enable high-throughput proteome characterization in addition to species determination from the identification of peptides distinctive to a species. Highlighted herein is an application of a peptide-centric approach to the identification and quantitation of species-specific peptide identifiers using an in silico exploration and an experimental mass spectrometry-based method. The application to microbial communities is addressed with an in silico analysis of an artificial complex community of 25 microorganisms.

Bacteria↗

PubMed2005

Monitoring food quality by microfluidic electrophoresis, gas chromatography, and mass spectrometry techniques: effects of aquaculture on the sea bass (Dicentrarchus labrax).

Monitoring food quality is a critical task for analytical chemistry and an important way to preserve human health. Fish is a valuable source of highly digestible proteins and contains large amounts of polyunsaturated fatty acids and fat-soluble vitamins. Since the world's wild fish stocks are limited, farmed fish is nowadays proposed as an alternative to consumers. It is now emerging that the fish muscle protein content is assuming great importance from an aquaculture perspective. Many data have been collected on the physiology and biochemistry of fish muscle, but few proteomic studies are available on farmed fish. Application of proteomics to aquaculture may play a key role in the development of new farming strategies. In this paper, a proteomic approach based on SDS-PAGE separation of proteins, in situ protein hydrolysis, de novo sequencing of peptides by MALDI and ESI MS(2), protein identification, and relative quantitation of protein by denaturing capillary electrophoresis was coupled with the determination of fatty acids and metal ions content by GM-MS and ICPMS in farmed and wild sea bass filet. Our results show that aquaculture could induce significant chemical and biochemical differences in fish muscle that may have an impact on food quality.

Amino Acid Sequence↗

PubMed2004

Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling.

A number of extracellular stimuli, including soluble cytokines and insoluble matrix factors, are known to influence murine embryonic stem cell self-renewal and differentiation behavioral responses via intracellular signaling pathways, but their net effects in combination are difficult to understand. To gain insight concerning key intracellular signals governing these behavioral responses, we employ a multivariate systems analysis of proteomic data generated from combinatorial stimulation of mouse embryonic stem cells by fibronectin, laminin, leukemia-inhibitory factor, and fibroblast growth factor 4. Phosphorylation states of 31 intracellular signaling network components were obtained across 16 different stimulus conditions at three time points by quantitative Western blotting, and partial-least-squares modeling was used to determine which components were most strongly correlated with cell proliferation and differentiation rate constants obtained from flow cytometry measurements of Oct-4 expression levels. This data-driven, multivariate (16 conditions x 31 components x 3 time points = approximately 1,500 values) proteomic approach identified a set of signaling network components most critically associated (positively or negatively) with differentiation (Stat3, Raf1, MEK, and ERK), proliferation of undifferentiated cells (MEK and ERK), and proliferation of differentiated cells (PKB alpha, Stat3, Src, and PKC epsilon). These predictions were found to be consistent with previous in vivo literature, along with direct in vitro test here by a peptide inhibitor of PKC epsilon. Our results demonstrate how a computational systems biology approach can elucidate key sets of intracellular signaling protein activities that combine to govern cell phenotypic responses to extracellular cues.

Animals↗

PubMed2002

Untangling the wires: a strategy to trace functional interactions in signaling and gene networks.

Emerging technologies have enabled the acquisition of large genomics and proteomics data sets. However, current methodologies for analysis do not permit interpretation of the data in ways that unravel cellular networking. We propose a quantitative method for determining functional interactions in cellular signaling and gene networks. It can be used to explore cell systems at a mechanistic level or applied within a "modular" framework, which dramatically decreases the number of variables to be assayed. This method is based on a mathematical derivation that demonstrates how the topology and strength of network connections can be retrieved from experimentally measured network responses to successive perturbations of all modules. Importantly, our analysis can reveal functional interactions even when the components of the system are not all known. Under these circumstances, some connections retrieved by the analysis will not be direct but correspond to the interaction routes through unidentified elements. The method is tested and illustrated by using computer-generated responses of a modeled mitogen-activated protein kinase cascade and gene network.

Animals↗

PubMed1999

Microfabricated modules for sample handling, sample concentration and flow mixing: application to protein analysis by tandem mass spectrometry.

The comprehensive analysis of biological systems requires a combination of genomic and proteomic efforts. The large-scale application of current genomic technologies provides complete genomic DNA sequences, sequence tags for expressed genes (EST's), and quantitative profiles of expressed genes at the mRNA level. In contrast, protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the proteins expressed by a tissue or cell. The sensitivity of protein analysis technology is primarily limited by the loss of analytes, due to adsorption to surfaces, and sample contamination during handling. Here we summarize our work on the development and use of microfabricated fluidic systems for the manipulation of minute amounts of peptides and delivery to an electrospray ionization tandem mass spectrometer. New data are also presented that further demonstrate the potential of these novel approaches. Specifically, we describe the use of microfabricated devices as modules to deliver femtomole amounts of protein digests to the mass spectrometer for protein identification. We also describe the use of a microfabricated module for the generation of solvent gradients at nl/min flow rates for gradient chromatography-tandem mass spectrometry. The use of microfabricated fluidic systems reduces the risk of sample contamination and sample loss due to adsorption to wetted surfaces. The ability to assemble dedicated modular systems and to operate them automatically makes the use of microfabricated systems attractive for the sensitive and large-scale analysis of proteins.

Equipment Design↗

PubMed2003

Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this "switching time" was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated "simultaneously" in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to approximately 80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm +/- 0.11 ppm (mean +/- confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams.

Amino Acid Sequence↗