Biomedical subjects

Leonard J Foster

Publications and source records attributed to Leonard J Foster.

15 recordsLinked to original sources

PubMed2026

Single-Cell Proteomics Reveals Proteome Remodeling and Cellular Heterogeneity During NGF-Induced PC12 Neuronal Differentiation.

Single-cell proteomics enables direct measurement of cellular heterogeneity during dynamic biological processes, but its application to fragile and highly adherent neuronal models remains challenging. Here, we developed and applied an optimized single-cell proteomics workflow to characterize proteome remodeling during nerve growth factor (NGF)-induced differentiation of PC12 cells. To enable reliable single-cell analysis, we implemented gentle dissociation, antiaggregation strategies, and thermal inkjet-based cell dispensing, achieving high accuracy in single-cell isolation. Inclusion of n-dodecyl-β-d-maltoside (DDM) improved recovery of membrane-associated and low-solubility proteins. Coupled with LC-ion mobility-mass spectrometry, this workflow enabled quantification of 2,000-3,000 proteins per cell across the differentiation time course. Single-cell proteomic analysis revealed progressive and heterogeneous proteome remodeling during differentiation. While undifferentiated cells formed a relatively homogeneous population, later stages (Days 4-6) exhibited increased variability, including multimodal protein abundance distributions and separation into distinct subpopulations. Dimensionality reduction, clustering, and non-negative matrix factorization identified multiple coexisting proteomic states within the same time points, reflecting asynchronous differentiation trajectories. These subpopulations were characterized by coordinated differences in pathways related to intracellular trafficking, protein translation, cytoskeletal organization, and neuronal maturation. Comparison with bulk proteomics demonstrated that proteins associated with differentiated neuronal states, including those involved in neurite formation and structural remodeling, are underrepresented in population-averaged measurements but are enriched within specific single-cell subpopulations. Temporal and cluster-resolved analyses further revealed distinct protein expression trajectories, including early decreases in cell cycle and metabolic pathways and later increases in neuronal structural and regulatory proteins. Together, this study establishes an optimized workflow for single-cell proteomics of neuronal systems and demonstrates that NGF-induced PC12 differentiation proceeds through heterogeneous and divergent proteomic states that are not resolved by bulk analysis.

Animals↗

PubMed2026

Toward large-scale mass spectrometry-based omics for clinical applications.

INTRODUCTION: As healthcare advances toward personalized medicine, mass spectrometry-based research is advancing our understanding of cellular biology and disease states, and translating these findings into clinical applications. This review highlights recent advances in methodology and technology that demonstrate the capabilities of mass spectrometry-based proteomics, lipidomics, and metabolomics in clinical practice. AREAS COVERED: The ability to directly analyze functional molecules with mass spectrometry uncovers crucial clinical information. Each data modality (proteins, lipids, and metabolites) provides essential insight into healthy and disease states. As technology advances, integrating data from different modalities unlocks new possibilities for clinical research. To gain the most from this multi-omic data, unsupervised integration methods can provide detailed insights into complex biological processes. As the field applies this knowledge, healthcare could experience significant leaps in the near future. This review examines recent advancements in mass spectrometry-based proteomics, lipidomics, and metabolomics, focusing on how improvements in sample preparation, automation, and multi-omics data integration are making large-scale clinical studies more accessible. EXPERT OPINION: Recent technical and methodological advancements in mass spectrometry analysis have propelled healthcare toward a tipping point, shifting from traditional RNA- and DNA-based research to downstream analysis of protein, lipid, and metabolite effectors.

Humans↗

PubMed2006

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.

Animals↗

PubMed2006

Quantitative comparison of caste differences in honeybee hemolymph.

The honeybee, Apis mellifera, is an invaluable partner in agriculture around the world both for its production of honey and, more importantly, for its role in pollination. Honeybees are largely unexplored at the molecular level despite a long and distinguished career as a model organism for understanding social behavior. Like other eusocial insects, honeybees can be divided into several castes: the queen (fertile female), workers (sterile females), and drones (males). Each caste has different energetic and metabolic requirements, and each differs in its susceptibility to pathogens, many of which have evolved to take advantage of the close social network inside a colony. Hemolymph, arthropods' equivalent to blood, distributes nutrients throughout the bee, and the immune components contained within it form one of the primary lines of defense against invading microorganisms. In this study we have applied qualitative and quantitative proteomics to gain a better understanding of honeybee hemolymph and how it varies among the castes and during development. We found large differences in hemolymph protein composition, especially between larval and adult stage bees and between male and female castes but even between adult workers and queens. We also provide experimental evidence for the expression of several unannotated honeybee genes and for the detection of biomarkers of a viral infection. Our data provide an initial molecular picture of honeybee hemolymph, to a greater depth than previous studies in other insects, and will pave the way for future biochemical studies of innate immunity in this animal.

Algorithms↗

PubMed2006

A mammalian organelle map by protein correlation profiling.

Protein localization to membrane-enclosed organelles is a central feature of cellular organization. Using protein correlation profiling, we have mapped 1,404 proteins to ten subcellular locations in mouse liver, and these correspond with enzymatic assays, marker protein profiles, and confocal microscopy. These localizations allowed assessment of the specificity in published organellar proteomic inventories and demonstrate multiple locations for 39% of all organellar proteins. Integration of proteomic and genomic data enabled us to identify networks of coexpressed genes, cis-regulatory motifs, and putative transcriptional regulators involved in organelle biogenesis. Our analysis ties biochemistry, cell biology, and genomics into a common framework for organelle analysis.

Animals↗

PubMed2006

Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver.

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver tissue sources. Indeed approximately one-third of the proteins identified in the soluble fractions are associated predominantly to one of the three tissues, indicating a tissue-dependent regulation of mitochondrial proteins. Furthermore a small percentage of the mitochondrial proteome is unique to each tissue.

Amino Acid Sequence↗

PubMed2006

Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC).

The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, alpha-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 +/- 0.5-fold by SILAC and 2.9 +/- 0.8-fold by immunoblotting. Insulin caused GLUT4 and alpha-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably alpha-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane.

Actinin↗

PubMed2006

Modification-specific proteomics of plasma membrane proteins: identification and characterization of glycosylphosphatidylinositol-anchored proteins released upon phospholipase D treatment.

Plasma membrane proteins are displayed through diverse mechanisms, including anchoring in the extracellular leaflet via glycosylphosphatidylinositol (GPI) molecules. GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family, and their importance is well-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural heterogeneity of the GPI moiety, making PLD a generally useful reagent for proteomic investigations of GPI-anchored proteins in a variety of cells, tissues, and organisms. A total of 11 human GPI-APs and 35 Arabidopsis thaliana GPI-APs were identified, representing a significant addition to the number of experimentally detected GPI-APs in both species. Computational GPI-AP sequence analysis tools were investigated for the characterization of the identified GPI-APs, and these demonstrated that there is some discrepancy in their efficiency in classification of GPI-APs and the exact assignment of omega-sites. This study highlights the efficiency of an integrative proteomics approach that combines experimental and computational methods to provide the selectivity, specificity, and sensitivity required for characterization of post-translationally modified membrane proteins.

Amino Acid Sequence↗

PubMed2005

Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation.

One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely useful for analyzing complex protein expression patterns and, when applied quantitatively, can be used to resolve subtle differences between samples. Thus, we used MS to characterize changes in expression of membrane protein markers before and after short-term induction of osteoblast (OB) differentiation in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and cytochemistry. Quantitative real-time reverse transcription-polymerase chain reaction confirmed the presence of mRNA of these membrane proteins. However, with the exception of ALP, no concordance was detected between the changes in levels of gene and protein expression during OB differentiation. In conclusion, MS-based proteomics can reveal novel markers for MSCs that can be used for their isolation and for monitoring OB differentiation.

Bone Marrow Cells↗

PubMed2004

RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers.

Focal adhesions are specialized attachment and signaling centers that form at sites of cell-matrix contacts. We employed a quantitative mass spectrometry-based method called SILAC to identify and quantify proteins interacting in an attachment-dependent manner with focal adhesion proteins. Subsequent confocal microscopy revealed a previously undescribed structure, which we have termed a spreading initiation center (SIC), existing only in early stages of cell spreading. SICs contain focal adhesion markers, appear to be surrounded by an actin sheath, and, surprisingly, contain numerous RNA binding proteins, ribosomal RNA, and perhaps other RNAs. Interfering with the function of FUS/TLS, hnRNP K, and hnRNP E1 results in increased spreading. Spreading initiation centers are ribonucleoprotein complexes distinct from focal adhesions and demonstrate a role for RNA and RNA binding proteins in the initiation of cell spreading.

Actins↗

PubMed2004

Intracellular delivery of phosphatidylinositol (3,4,5)-trisphosphate causes incorporation of glucose transporter 4 into the plasma membrane of muscle and fat cells without increasing glucose uptake.

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P(3)) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P(3) intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P(3) caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phospho-Akt and GLUT4. Intracellular delivery of PI(3,4,5)P(3) using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P(3) was not reproduced by carrier-PI 4,5-bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P(3) elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P(3) did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of exogenously delivered PI(3,4,5)P(3) to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport.

3T3-L1 Cells↗

PubMed2003

Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root development. We here present a general mass spectrometry-based proteomic "shave-and-conquer" strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date.

Amino Acid Sequence↗

PubMed2003

Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.

Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.

Cell Fractionation↗

PubMed2003

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

Epidermal Growth Factor↗

PubMed2003

Mass spectrometric-based approaches in quantitative proteomics.

Classically, experiments aimed at studying changes in protein expression have always followed a small set of proteins. This focused approach was necessary since tools to efficiently analyze large numbers of proteins were simply not available. Large-scale quantitative proteomics promises to produce reams of data that previously would have taken decades to measure with classical methods. Mass spectrometry is already a well-established protein identification tool and recent methodological developments indicate that it can also be successfully applied to extract quantitative data of protein abundance. From the first reports 4 years ago, numerous schemes to take advantage of stable isotope nuclei incorporation in proteins and peptides have been developed. Here we review the benefits and pitfalls of some of the most commonly used protocols, focusing on a procedure now being used extensively in our laboratory, stable isotope labeling with amino acids in cell culture (SILAC). The basic theory, application, and data analysis of a SILAC experiment are discussed. The emerging nature of these techniques and the rapid pace of technological development make forecasting the directions of the field difficult but we speculate that SILAC will soon be a key tool of quantitative proteomics.

Amino Acids↗