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1.  Glycogen Synthase Kinase (GSK) 3β Phosphorylates and Protects Nuclear Myosin 1c from Proteasome-Mediated Degradation to Activate rDNA Transcription in Early G1 Cells 
PLoS Genetics  2014;10(6):e1004390.
Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β−/− mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation.
Author Summary
Nuclear actin and myosin are essential regulators of gene expression. At the exit of mitosis, nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by modulating assembly of the chromatin remodeling complex WICH with the subunits WSTF and SNF2h and, crucially, facilitating H3K9 acetylation by the histone acetyl transferase PCAF. The molecular mechanism by which NM1 is regulated remains however unknown. Here, we conducted a genome-wide screen and demonstrate that GSK3β is selectively coupled to the rDNA transcription unit. In embryonic fibroblasts lacking GSK3β there is a significant drop in rRNA synthesis levels and the rDNA is devoid of actin, NM1 and SNF2h. Concomitantly with a transcriptional block we reveal decreased levels of histone H3 acetylation by the histone acetyl transferase PCAF. At G1, transcriptional repression in the GSK3β knockout mouse embryonic fibroblasts, leads to NM1 ubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β suppresses NM1 degradation through the ubiquitin-proteasome system, facilitates NM1 association with the rDNA chromatin and transcription activation at G1. We therefore propose a novel and fundamental role for GSK3β as essential regulator of rRNA synthesis and cell cycle progression.
PMCID: PMC4046919  PMID: 24901984
2.  Dysregulation of ubiquitin homeostasis and β-catenin signaling promote spinal muscular atrophy 
The Journal of Clinical Investigation  2014;124(4):1821-1834.
The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. Here, we determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, we observed widespread perturbations in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1). SMN physically interacted with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways led to β-catenin accumulation, and pharmacological inhibition of β-catenin robustly ameliorated neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of β-catenin was restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of β-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. Our data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent β-catenin signaling, highlighting ubiquitin homeostasis and β-catenin as potential therapeutic targets for SMA.
PMCID: PMC3973095  PMID: 24590288
3.  Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy 
Genome Medicine  2013;5(10):95.
Spinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations in the survival motor neuron 1 (SMN1) gene. Recent breakthroughs in preclinical research have highlighted several potential novel therapies for SMA, increasing the need for robust and sensitive clinical trial platforms for evaluating their effectiveness in human patient cohorts. Given that most clinical trials for SMA are likely to involve young children, there is a need for validated molecular biomarkers to assist with monitoring disease progression and establishing the effectiveness of therapies being tested. Proteomics technologies have recently been highlighted as a potentially powerful tool for such biomarker discovery.
We utilized label-free proteomics to identify individual proteins in pathologically-affected skeletal muscle from SMA mice that report directly on disease status. Quantitative fluorescent western blotting was then used to assess whether protein biomarkers were robustly changed in muscle, skin and blood from another mouse model of SMA, as well as in a small cohort of human SMA patient muscle biopsies.
By comparing the protein composition of skeletal muscle in SMA mice at a pre-symptomatic time-point with the muscle proteome at a late-symptomatic time-point we identified increased expression of both Calreticulin and GRP75/Mortalin as robust indicators of disease progression in SMA mice. We report that these protein biomarkers were consistently modified in different mouse models of SMA, as well as across multiple skeletal muscles, and were also measurable in skin biopsies. Furthermore, Calreticulin and GRP75/Mortalin were measurable in muscle biopsy samples from human SMA patients.
We conclude that label-free proteomics technology provides a powerful platform for biomarker identification in SMA, revealing Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers capable of reporting on disease progression in samples of muscle and skin.
PMCID: PMC3979019  PMID: 24134804
4.  The Salmonella Kinase SteC Targets the MAP Kinase MEK to Regulate the Host Actin Cytoskeleton 
Cell Host & Microbe  2012;12(5):657-668.
After host cell entry, Salmonella replicate in membrane-bound compartments, which accumulate a dense meshwork of F-actin through the kinase activity of the Salmonella SPI-2 type III secretion effector SteC. We find that SteC promotes actin cytoskeleton reorganization by activating a signaling pathway involving the MAP kinases MEK and ERK, myosin light chain kinase (MLCK) and Myosin IIB. Specifically, SteC phosphorylates MEK directly on serine 200 (S200), a previously unstudied phosphorylation site. S200 phosphorylation is predicted to displace a negative regulatory helix causing autophosphorylation on the known MEK activatory residues, S218 and S222. In support of this, substitution of S200 with alanine prevented phosphorylation on S218 and S222, and phosphomimetic mutations of S200 stimulated phosphorylation of these residues. Both steC-null and kinase-deficient mutant strains displayed enhanced replication in infected cells, suggesting that SteC manipulates the actin cytoskeleton to restrain bacterial growth, thereby regulating virulence.
Graphical Abstract
► Salmonella SteC reorganizes F-actin via a MEK/ERK/MLCK/Myosin IIB pathway ► SteC phosphorylates MEK1 S200, which is required for SteC-induced MEK activation ► MEK1 S200 phosphorylation induces autophosphorylation on residues S218/222 ► SteC also controls bacterial growth via its kinase function
PMCID: PMC3510437  PMID: 23159055
5.  Combining Comparative Proteomics and Molecular Genetics Uncovers Regulators of Synaptic and Axonal Stability and Degeneration In Vivo 
PLoS Genetics  2012;8(8):e1002936.
Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel “top-down” approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.
Author Summary
In diseases affecting the nervous system, such as Alzheimer's disease and motor neuron disease, the breakdown of synaptic connections between neurons is a critical early event, contributing to disease onset and progression. However, we still know very little about the molecular machinery present in synaptic and axonal compartments of neurons that regulate their stability and cause breakdown during neurodegeneration. In this study we examined the protein composition of healthy and degenerating synapse-enriched fractions isolated from the brains of mice in order to identify early molecular changes occurring during neurodegeneration. We identified a range of proteins and cellular pathways that were modulated in synapse-enriched fractions during the early phases of degeneration, many of which were already known to regulate synaptic function. Similar molecular alterations were found in synapse-enriched fractions prepared from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. Data from these proteomic studies were then used to design experiments in Drosophila, in which we found that at least six of the individual proteins modified in degenerating synapses from mice were capable of independently regulating neuronal stability and degeneration in vivo. Designing novel therapeutics to target these proteins and pathways may help to delay or prevent neurodegeneration across a range of diseases.
PMCID: PMC3431337  PMID: 22952455
6.  SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy 
Human Molecular Genetics  2010;19(21):4216-4228.
Reduced expression of the survival motor neuron (SMN) gene causes the childhood motor neuron disease spinal muscular atrophy (SMA). Low levels of ubiquitously expressed SMN protein result in the degeneration of lower motor neurons, but it remains unclear whether other regions of the nervous system are also affected. Here we show that reduced levels of SMN lead to impaired perinatal brain development in a mouse model of severe SMA. Regionally selective changes in brain morphology were apparent in areas normally associated with higher SMN levels in the healthy postnatal brain, including the hippocampus, and were associated with decreased cell density, reduced cell proliferation and impaired hippocampal neurogenesis. A comparative proteomics analysis of the hippocampus from SMA and wild-type littermate mice revealed widespread modifications in expression levels of proteins regulating cellular proliferation, migration and development when SMN levels were reduced. This study reveals novel roles for SMN protein in brain development and maintenance and provides the first insights into cellular and molecular pathways disrupted in the brain in a severe form of SMA.
PMCID: PMC2951867  PMID: 20705736
7.  A Quantitative Spatial Proteomics Analysis of Proteome Turnover in Human Cells* 
Molecular & Cellular Proteomics : MCP  2011;11(3):M111.011429.
Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.
PMCID: PMC3316722  PMID: 21937730
8.  Application of electrospray mass spectrometry to the structural determination of glycosylphosphatidylinositol membrane anchors 
Glycobiology  2010;20(5):576-585.
The addition of glycosylphosphatidylinositol (GPI) anchors to proteins is an important posttranslational modification in eukaryotic cells. The complete structural elucidation of GPI anchors is a complex process that requires relatively large amounts of starting material. In this paper, we assess the degree of structural information that can be obtained by applying electrospray mass spectrometry and tandem mass spectrometry to permethylated GPI glycans prepared from a well-characterized GPI-anchored glycoprotein, the variant surface glycoprotein from Trypanosoma brucei. All GPI glycans contain a non-N-acetylated glucosamine residue, and permethylation leads to the formation of a fixed positive charge on the glycans, in the form of a quaternary amine. The permethylated glycans were detected as [M +- Na]2+- ions, and tandem mass spectrometry of these ions produced substantial, albeit incomplete, structural information on the branching patterns and linkage types for various GPI glycoforms of the variant surface glycoprotein.
PMCID: PMC2850939  PMID: 20100693
glycosylphosphatidylinositol; GPI anchor; mass spectrometry; Trypanosoma brucei; variant surface glycoprotein
9.  A Quantitative Proteomics Analysis of Subcellular Proteome Localization and Changes Induced by DNA Damage* 
A major challenge in cell biology is to identify the subcellular distribution of proteins within cells and to characterize how protein localization changes under different cell growth conditions and in response to stress and other external signals. Protein localization is usually determined either by microscopy or by using cell fractionation combined with protein blotting techniques. Both these approaches are intrinsically low throughput and limited to the analysis of known components. Here we use mass spectrometry-based proteomics to provide an unbiased, quantitative, and high throughput approach for measuring the subcellular distribution of the proteome, termed “spatial proteomics.” The spatial proteomics method analyzes a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass-labeled with heavy isotopes. This was used here to measure the relative distribution between cytoplasm, nucleus, and nucleolus of over 2,000 proteins in HCT116 cells. The data show that, at steady state, the proteome is predominantly partitioned into specific subcellular locations with only a minor subset of proteins equally distributed between two or more compartments. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations, shown here by characterizing dynamic changes in protein localization elicited during the cellular response to DNA damage following treatment of HCT116 cells with etoposide. DNA damage was found to cause dissociation of the proteasome from inhibitory proteins and assembly chaperones in the cytoplasm and relocation to associate with proteasome activators in the nucleus.
PMCID: PMC2849709  PMID: 20026476
10.  Identification and Specific Localization of Tyrosine-Phosphorylated Proteins in Trypanosoma brucei▿ †  
Eukaryotic Cell  2009;8(4):617-626.
Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in T. brucei, a principle well known from higher eukaryotes.
PMCID: PMC2669198  PMID: 19181871
11.  Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei▿ † ‡  
Eukaryotic Cell  2009;8(9):1407-1417.
A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB3H4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB3H4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.
PMCID: PMC2747833  PMID: 19633269
12.  The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness 
The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.
PMCID: PMC2716717  PMID: 19346560
13.  Implications of childhood obesity for adult health: findings from thousand families cohort study 
BMJ : British Medical Journal  2001;323(7324):1280-1284.
To determine whether being overweight in childhood increases adult obesity and risk of disease.
Prospective cohort study.
City of Newcastle upon Tyne.
932 members of thousand families 1947 birth cohort, of whom 412 attended for clinical examination age 50.
Main outcome measures
Blood pressure; carotid artery intima-media thickness; fibrinogen concentration; total, low density lipoprotein, and high density lipoprotein cholesterol concentrations; triglyceride concentration; fasting insulin and 2 hour glucose concentrations; body mass index; and percentage body fat.
Body mass index at age 9 years was significantly correlated with body mass index age 50 (r=0.24, P<0.001) but not with percentage body fat age 50 (r=0.10, P=0.07). After adult body mass index had been adjusted for, body mass index at age 9 showed a significant inverse association with measures of lipid and glucose metabolism in both sexes and with blood pressure in women. However, after adjustment for adult percentage fat instead of body mass index, only the inverse associations with triglycerides (regression coefficient= −0.21, P<0.01) and total cholesterol (−0.17, P<0.05) in women remained significant.
Little tracking from childhood overweight to adulthood obesity was found when using a measure of fatness that was independent of build. Only children who were obese at 13 showed an increased risk of obesity as adults. No excess adult health risk from childhood or teenage overweight was found. Being thin in childhood offered no protection against adult fatness, and the thinnest children tended to have the highest adult risk at every level of adult obesity.
What is already known on this topicMany studies have found that body mass index in childhood is significantly correlated with body mass index in adulthoodObese children have been found to have higher all cause mortality as adultsWhat this study addsNo excess health risk from childhood overweight was foundChildhood body mass index was linked to adulthood body mass index but not percentage body fatOnly children who were obese at 13 showed a significant increased risk of obesity as adultsPeople who were thinnest as children and fattest as adults tended to have the highest adult risk
PMCID: PMC60301  PMID: 11731390
14.  Risk of cardiovascular disease measured by carotid intima-media thickness at age 49-51: lifecourse study 
BMJ : British Medical Journal  2000;320(7230):273-278.
To quantify the direct and indirect effects of fetal life, childhood, and adult life on risk of cardiovascular disease at age 49-51 years.
Follow up study of the “Newcastle thousand families” birth cohort established in 1947.
154 men and 193 women who completed a health and lifestyle questionnaire and attended for clinical examination between October 1996 and December 1998.
Main outcome measures
Correlations between mean intima-media thickness of the carotid artery (carotid intima-media thickness) and family history, birth weight, and socioeconomic position around birth; socioeconomic position, growth, illness, and adverse life events in childhood; and adult socioeconomic position, lifestyle, and biological risk markers. Proportions of variance in carotid intima-media thickness that were accounted for by each stage of the lifecourse.
Socioeconomic position at birth and birth weight were negatively associated with carotid intima-media thickness, although only social class at birth in women was a statistically significant covariate independent of adult lifestyle. These early life variables accounted directly for 2.2% of total variance in men and 2.0% in women. More variation in carotid intima-media thickness was explained by adult socioeconomic position and lifestyle, which accounted directly and indirectly for 3.4% of variance in men (95% confidence interval 0.5% to 6.2%) and 7.6% in women (2.1% to 13.0%). Biological risk markers measured in adulthood independently accounted for a further 9.5% of variance in men (2.4% to 14.2%) and 4.9% in women (1.6% to 7.4%).
Adult lifestyle and biological risk markers were the most important determinants of the cardiovascular health of the study members of the Newcastle thousand families cohort at age 49-51 years. The limited overall effect of early life factors may reflect the postwar birth year of this cohort.
PMCID: PMC27272  PMID: 10650022
15.  Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO2 and Ti-IMAC 
Analytical Chemistry  2014;86(20):10296-10302.
Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, “well-to-well” quantitative reproducibility indistinguishable to internal SILAC standards, and robust “plate-to-plate” reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.
PMCID: PMC4206527  PMID: 25233145

Results 1-15 (15)