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2.  Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium* 
The Journal of Biological Chemistry  2014;289(13):8818-8827.
Background: Myocardial infarction (MI) leads to proteolytic cleavage of cMyBP-C (hC0C1f) and decreased contractility.
Results: hC0C1f can incorporate into the human cardiac sarcomere, depressing force generation and increasing tension cost.
Conclusion: Interaction between hC0C1f and both actin and α-tropomyosin causes disruption of intact cMyBP-C function.
Significance: Proteolytic cleavage of cMyBP-C is sufficient to cause contractile dysfunction following MI.
Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca2+ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca2+ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.
doi:10.1074/jbc.M113.541128
PMCID: PMC3979389  PMID: 24509847
Contractile Protein; Heart Failure; Myocardial Infarction; Protein Degradation; Protein-Protein Interactions
3.  Top-down Targeted Proteomics for Deep Sequencing of Tropomyosin Isoforms 
Journal of proteome research  2012;12(1):187-198.
SUMMARY
Tropomyosins (Tm) constitute a family of ubiquitous and highly conserved actin-binding proteins, playing essential roles in a variety of biological processes. Tm isoforms produced by multiple Tm encoding genes and alternatively expressed exons along with post-translational modifications (PTMs) regulate Tm function. Therefore, to gain a better understanding of the functional role of Tm, it is essential to fully characterize Tm isoforms. Herein, we developed a top-down high-resolution mass spectrometry (MS) based targeted proteomics method for comprehensive characterization of Tm isoforms. α–Tm was identified to be the predominant isoform in swine cardiac muscle. We further characterized its sequence and localized the PTMs such as acetylation and phosphorylation as well as amino acid polymorphisms. Interestingly, we discovered a “novel” Tm isoform that does not match with any of the currently available swine Tm sequences. A deep sequencing of this isoform by top-down MS revealed an exact match with mouse β–Tm sequence, suggesting that this “novel” isoform is swine β–Tm which is 100% conserved between swine and mouse. Taken together, we demonstrated that top-down targeted proteomics provides a powerful tool for deep sequencing of Tm isoforms from genetic variations together with complete mapping of the PTM sites.
doi:10.1021/pr301054n
PMCID: PMC3596867  PMID: 23256820
Tropomyosin; Muscle Contraction; Actin Filament; Mass Spectrometry; Electron Capture Dissociation
4.  Value of multidetector computed tomography in the diagnosis of mucosa-associated lymphoid tissue-lymphomas in the parotid gland: A case report and review of the literature 
Oncology Letters  2013;7(3):781-786.
The present study aimed to review the multidetector computed tomography (MDCT) imaging features of eight mucosa-associated lymphoid tissue (MALT)-lymphoma cases of the parotid gland and to explore the diagnostic value of MDCT. A total of eight patients with pathologically confirmed MALT-lymphomas of the parotid gland underwent pre-operative MDCT plain and dual-phase scans. The changes in the CT values and enhancement patterns of the tumors were assessed. Quantitative analysis was performed to determine the CT value changes of the tumors in the various enhanced phases compared with the plain scan. The MALT-lymphomas of the parotid gland exhibited even density isodense or hyperdense nodules, with occasional calcification and necrosis. The dual-phase scan of the MALT-lymphomas revealed a pattern of lower or moderate enhancement, circumambient enhancement or delayed enhancement. The MALT-lymphomas were closely associated with Sjögre’s syndrome and demonstrated malignant features and isodense or hyperdense nodules and lower or moderate enhancement on the CT scans.
doi:10.3892/ol.2013.1769
PMCID: PMC3919924  PMID: 24520295
parotid gland; mucosa-associated lymphoid tissue lymphomas; computer tomography; enhancement
5.  HNO, Redox Switches, Cardiac Myofilaments, and Heart Failure: A Prequel to Novel Therapeutics? 
Circulation research  2012;111(8):954-956.
doi:10.1161/CIRCRESAHA.112.278416
PMCID: PMC3494458  PMID: 23023505
heart failure; contractility; nitroxyl; disulfide; mass spectrometry
6.  The role of industrial nitrogen in the global nitrogen biogeochemical cycle 
Scientific Reports  2013;3:2579.
Haber-Bosch nitrogen (N) has been increasingly used in industrial products, e.g., nylon, besides fertilizer. Massive numbers of species of industrial reactive N (Nr) have emerged and produced definite consequences but receive little notice. Based on a comprehensive inventory, we show that (1) the industrial N flux has increased globally from 2.5 to 25.4 Tg N yr−1 from 1960 through 2008, comparable to the NOx emissions from fossil fuel combustion; (2) more than 25% of industrial products (primarily structural forms, e.g., nylon) tend to accumulate in human settlements due to their long service lives; (3) emerging Nr species define new N-assimilation and decomposition pathways and change the way that Nr is released to the environment; and (4) the loss of these Nr species to the environment has significant negative human and ecosystem impacts. Incorporating industrial Nr into urban environmental and biogeochemical models could help to advance urban ecology and environmental sciences.
doi:10.1038/srep02579
PMCID: PMC3759834  PMID: 23999540
7.  CistromeMap: a knowledgebase and web server for ChIP-Seq and DNase-Seq studies in mouse and human 
Bioinformatics  2012;28(10):1411-1412.
Summary: Transcription and chromatin regulators, and histone modifications play essential roles in gene expression regulation. We have created CistromeMap as a web server to provide a comprehensive knowledgebase of all of the publicly available ChIP-Seq and DNase-Seq data in mouse and human. We have also manually curated metadata to ensure annotation consistency, and developed a user-friendly display matrix for quick navigation and retrieval of data for specific factors, cells and papers. Finally, we provide users with summary statistics of ChIP-Seq and DNase-Seq studies.
Availability: Freely available on the web at http://cistrome.dfci.harvard.edu/pc/
Contact: yzhang@tongji.edu.cn; xsliu@jimmy.harvard.edu
doi:10.1093/bioinformatics/bts157
PMCID: PMC3348563  PMID: 22495751
8.  The vasodilatory effect of testosterone on renal afferent arterioles 
Gender Medicine  2012;9(2):103-111.
Background
Gender differences exist in a variety of cardiovascular and renal diseases, and testosterone may contribute to the discrepancy. Afferent arterioles (Af-Art) are the major resistance vessels in the kidney, and play an important role in the development of renal injury and hypertension.
Objective
The present study aimed to determine the acute effect and underlying mechanism(s) of testosterone on Af-Art.
Methods
The mRNA expression of androgen receptors (AR) in microdissected Af-Art was measured by RT-PCR. An in vitro microperfusion model was used to measure the diameter of Ar-Art in mice. Nitric oxide (NO) was evaluated by an NO-sensitive fluorescent dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate.
Results
Testosterone had no effect on microperfused Af-Art when added into the bath. Therefore we pre-constricted the Af-Art to about 30% with norepinephrine (NE, 10−6mol/L); administration of testosterone (10−9 to 10−7mol/L) subsequently dilated the Af-Art in a dose-dependent manner (p<0.001; n=7). AR mRNA was expressed in microdissected Af-Art measured by RT-PCR. An AR antagonist, flutamide (10−5mol/L), totally blocked testosterone (10−8mol/L)-induced vasodilator effect. NO production of Af-Art wall was increased when testosterone was added into the bath solution after NE treatment, from 278.4 ± 12.1 units/min to 351.2 ± 33.1 units/min (p<0.05; n=3). In the presence of NO inhibition with NG-nitro-L-arginine methyl ester (L-NAME, 3×10−4mol/L), the testosterone-induced dilatation was blunted compared with NE (p<0.05).
Conclusions
We conclude that testosterone dilates pre-constricted mouse Af-Art in a dose-dependent manner by activation of AR and partially mediated by NO.
doi:10.1016/j.genm.2012.02.003
PMCID: PMC3322301  PMID: 22445695
kidney; testosterone; androgen receptors; nitric oxide
9.  The Role of Community Mixing Styles in Shaping Epidemic Behaviors in Weighted Networks 
PLoS ONE  2013;8(2):e57100.
The dynamics of infectious diseases that are spread through direct contact have been proven to depend on the strength of community structure or modularity within the underlying network. It has been recently shown that weighted networks with similar modularity values may exhibit different mixing styles regarding the number of connections among communities and their respective weights. However, the effect of mixing style on epidemic behavior was still unclear. In this paper, we simulate the spread of disease within networks with different mixing styles: a dense-weak style (i.e., many edges among the communities with small weights) and a sparse-strong style (i.e., a few edges among the communities with large weights). Simulation results show that, with the same modularity: 1) the mixing style significantly influences the epidemic size, speed, pattern and immunization strategy; 2) the increase of the number of communities amplifies the effect of the mixing style; 3) when the mixing style changes from sparse-strong to dense-weak, there is a ‘saturation point’, after which the epidemic size and pattern become stable. We also provide a mean-field solution of the epidemic threshold and size on weighted community networks with arbitrary external and internal degree distribution. The solution explains the effect of the second moment of the degree distribution, and a symmetric effect of internal and external connections (incl. degree distribution and weight). Our study has both potential significance for designing more accurate metrics for the community structure and exploring diffusion dynamics on metapopulation networks.
doi:10.1371/journal.pone.0057100
PMCID: PMC3577779  PMID: 23437321
10.  Comprehensive Analysis of Protein Modifications by Top-down Mass Spectrometry 
Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research.
doi:10.1161/CIRCGENETICS.110.957829
PMCID: PMC3320739  PMID: 22187450
Cardiovascular diseases; Proteomics; Electron Capture dissociation; Post-translational modification; Top-Down Mass Spectrometry
11.  A comparative study of 22-channel water-perfusion system and solid-state system with 36-sensors in esophageal manometery 
BMC Gastroenterology  2012;12:157.
Background
To compare the characteristics between 22-channel water-perfusion manometry (WPM) and solid-state manometry (SSM) with 36 sensors of the pressure measurements, as well as patients’ discomfort indices in nose and pharynx, the preparation and operation time of the manometry.
Methods
12 volunteers were included in the study. Each of the volunteers underwent esophageal manometry by both 22-channel water-perfusion catheter (WPC) and solid-state catheter (SSC) with 36 sensors in random order, and separated by 30 min. The subjects gave a VAS score soon after each test. Non-parametric tests were used to analyze the differences and Bland-Altman plots were used to assess the consistency of the two systems.
Results
During the wet swallows, there were significant differences between the two systems in three measurements of location of lower esophageal sphincter (LES) upper margin (Z = -2.11, P = 0.035), LES relax ratio (Z = -2.20, P = 0.028) and IRP4s (Z = -2.05, P = 0.041). During the jelly pocket swallows, LES relax ratio measurements of the two systems showed significant differences (Z = -2.805, P = 0.005). Further Bland–Altman plots analysis presented good agreement between the two systems measurements of location of LES upper margin, LES relax ratio and IRP4s. The discomfort indices of subjects’ nasal sensation were higher when inserting the solid-state catheter [5(3.75-5)] than water-perfusion one (2.5(2-4)) (Z = -2.471, P = 0.013), as well as the discomfort indices of pharyngeal sensation (7.5(4.75-9) vs. 4.5(3.75-6.5)), (Z = -2.354, P = 0.019). The preparation time for WPC was 40(39-41) minutes, which was much longer than that for SSC 32.5(31.75-33) minutes, (Z = -3.087, P = 0.002). And the nurses reported it’s much easier to insert WPC (Z = -3.126, P = 0.002).
Conclusions
In conclusion, most pressure measurements were consistent between WPM and SSM. Patients tolerated better with WPC, while for operators, the SSC presented more convenient.
doi:10.1186/1471-230X-12-157
PMCID: PMC3503791  PMID: 23134719
22-channel water-perfusion manometry; Solid-state manometry (SSM) with 36 sensors; Pressure measurements; Patients’ tolerance; Operators’ convenience; Comparative study
12.  Esophageal intraluminal baseline impedance is associated with severity of acid reflux and epithelial structural abnormalities in patients with gastroesophageal reflux disease 
Journal of Gastroenterology  2012;48(5):601-610.
Background
The esophageal intraluminal baseline impedance may be used to evaluate the status of mucosa integrity. Esophageal acid exposure decreases the baseline impedance. We aimed to compare baseline impedance in patients with various reflux events and with different acid-related parameters, and investigate the relationships between epithelial histopathologic abnormalities and baseline impedance.
Methods
A total of 229 GERD patients and 34 controls underwent 24-h multichannel intraluminal impedance and pH monitoring (MII–pH monitoring), gastroendoscopy, and completed a GERD questionnaire (GerdQ). We quantified epithelial intercellular spaces (ICSs) and expression of tight junction (TJ) proteins by histologic techniques.
Results
Mean baseline values in reflux esophagitis (RE) (1752 ± 1018 Ω) and non-erosive reflux disease (NERD) (2640 ± 1143 Ω) were significantly lower than in controls (3360 ± 1258 Ω; p < 0.001 and p = 0.001, respectively). Among NERD subgroups, mean baselines in the acid reflux group (2510 ± 1239 Ω) and mixed acid/weakly acidic reflux group (2393 ± 1009 Ω) were much lower than in controls (3360 ± 1258 Ω; p = 0.020 and p < 0.001, respectively). The mean baseline in severe RE patients was significantly lower than in mild RE patients (LA-C/D vs. LA-A/B: 970 ± 505 Ω vs. 1921 ± 1024 Ω, p < 0.001). There was a significant negative correlation between baseline value and acid exposure time (AET) (r = −0.41, p < 0.001), and a weak but significant correlation (r = −0.20, p = 0.007) between baseline value and weakly AET. Negative correlations were observed between ICS and the baseline impedance (r = −0.637, p < 0.001) and claudin-1 and the baseline impedance (r = −0.648, p < 0.001).
Conclusions
Patients with dominant acid reflux events and with longer AET have low baseline impedance. Baseline values are correlated with esophageal mucosal histopathologic changes such as dilated ICS and TJ alteration.
Electronic supplementary material
The online version of this article (doi:10.1007/s00535-012-0689-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00535-012-0689-6
PMCID: PMC3654188  PMID: 23076541
Baseline impedance; Acid reflux; Intercellular spaces; Tight junction
13.  Top-Down Quantitative Proteomics Identified Phosphorylation of Cardiac Troponin I as a Candidate Biomarker for Chronic Heart Failure 
Journal of proteome research  2011;10(9):4054-4065.
The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We have systematically analyzed thirty-six clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%Ptotal) were 56.4±3.5%, 36.9±1.6%, 6.1±2.4%, and 1.0±0.6% for postmortem hearts with normal cardiac function (n=7), early-stage of mild hypertrophy (n=5), severe hypertrophy/dilation (n=4), and end-stage CHF (n=6), respectively. In fresh transplant samples, the %Ptotal of cTnI from non-failing donor (n=4), and end-stage failing hearts (n=10) were 49.5±5.9% and 18.8±2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTM as disease biomarkers.
doi:10.1021/pr200258m
PMCID: PMC3170873  PMID: 21751783
Heart failure; Phosphorylation; Quantitative Proteomics; Top-Down Mass Spectrometry; Post-translational Modification; Cardiac troponin I
14.  In Vivo Phosphorylation Site Mapping in Mouse Cardiac Troponin I by High Resolution Top-Down Electron Capture Dissociation Mass Spectrometry: Ser22/23 Are the Only Sites Basally Phosphorylated† 
Biochemistry  2009;48(34):8161-8170.
Cardiac troponin I (cTnI) is the inhibitory subunit of cardiac troponin, a key myofilament regulatory protein complex located on the thin filaments of the contractile apparatus. cTnI is uniquely specific for the heart and is widely used in clinics as a serum biomarker for cardiac injury. Phosphorylation of cTnI plays a critical role in modulating cardiac function. cTnI is known to be regulated by protein kinase A and protein kinase C at five sites, Ser22/Ser23, Ser42/44, and Thr143, primarily based on results from in vitro phosphorylation assays by the specific kinase(s). However, a comprehensive characterization of phosphorylation of mouse cTnI occurring in vivo has been lacking. Herein, we have employed top-down mass spectrometry (MS) methodology with electron capture dissociation for precise mapping of in vivo phosphorylation sites of cTnI affinity purified from wild-type and transgenic mouse hearts. As demonstrated, top-down MS (analysis of intact proteins) is an extremely valuable technology for global characterization of labile phosphorylation occurring in vivo without a priori knowledge. Our top-down MS data unambiguously identified Ser22/23 as the only two sites basally phosphorylated in wild-type mouse cTnI with full sequence coverage, which was confirmed by the lack of phosphorylation in cTnI-Ala2 transgenic mice where Ser22/23 in cTnI have been rendered nonphosphorylatable by mutation to alanine.
doi:10.1021/bi900739f
PMCID: PMC3341416  PMID: 19637843
15.  The Protein Partners of GTP Cyclohydrolase I in Rat Organs 
PLoS ONE  2012;7(3):e33991.
Objective
GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat.
Methods and Results
A pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria.
Conclusion
GCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis.
doi:10.1371/journal.pone.0033991
PMCID: PMC3313957  PMID: 22479495
16.  Phosphorylation, but Not Alternative Splicing or Proteolytic Degradation, Is Conserved in Human and Mouse Cardiac Troponin T 
Biochemistry  2011;50(27):6081-6092.
Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca2+-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.
doi:10.1021/bi2006256
PMCID: PMC3312388  PMID: 21639091
17.  Single amino acid sequence polymorphisms in rat cardiac troponin revealed by top-down tandem mass spectrometry 
Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. cTn heterotrimers were affinity purified, desalted and then directly subjected to mass spectrometry using a 7 Tesla Thermo LTQ-FT-ICR instrument equipped with an ESI source. Molecular ions for N-terminally processed and acetylated cTnI and cTnT were readily detected as were other post-translationally modified forms of these proteins. cTnI was phosphorylated with a distribution of un-, mono- and bisphosphorylated forms of 41 ± 3%, 46 ± 1%, 13 ± 3%, respectively. cTnT was predominantly mono-phosphorylated and partially proteolyzed at the Glu29-Pro30 peptide bond. Also observed in high resolution spectra were ‘shadow’ peaks of similar intensity to ‘parent’ peaks exhibiting masses of cTnI+16 Da and cTnT+128 Da, subsequently shown by tandem mass spectrometry (MS/MS) to be single amino acid polymorphisms. Intact and protease-digested cTn subunits were fragmented by electron capture dissociation or collision activated dissociation to localize an Ala/Ser polymorphism at residue 7 of cTnI. Similar analysis of cTnT localized an additional Gln within a three residue alternative splice site beginning at residue 192. Besides being able to provide unique insights into the global state of post-translational modification of cTn subunits, high resolution top-down mass spectrometry readily revealed naturally occurring single amino acid sequence variants including a genetic polymorphism at residue 7 in cTnI, and an alternative splice isoform that affects a putative hinge region around residue 192 of cTnT, all of which co-exist within a single rat heart.
doi:10.1007/s10974-009-9168-y
PMCID: PMC3312389  PMID: 19165611
Phosphorylation; Alternative splicing; Proteomics; Post-translational modification; Electron capture dissociation
18.  Effective Enrichment and Mass Spectrometry Analysis of Phosphopeptides Using Mesoporous Metal Oxide Nanomaterials 
Analytical chemistry  2010;82(17):7193-7201.
Mass spectrometry (MS)-based phosphoproteomics remains challenging due to the low abundance of phosphoproteins and substoichiometric phosphorylation. This demands better methods to effectively enrich phosphoproteins/peptides prior to MS analysis. We have previously communicated the first use of mesoporous zirconium oxide (ZrO2) nanomaterials for effective phosphopeptide enrichment. Here we present the full report including the synthesis, characterization, and application of mesoporous titanium dioxide (TiO2), ZrO2, and hafnium oxide (HfO2) in phosphopeptide enrichment and MS analysis. Mesoporous ZrO2 and HfO2 are demonstrated to be superior to TiO2 for phosphopeptide enrichment from a complex mixture with high specificity (>99%), which could almost be considered as “a purification”, mainly because of the extremely large active surface area of mesoporous nanomaterials. A single enrichment and Fourier transform MS analysis of phosphopeptides digested from a complex mixture containing 7% of α-casein identified 21 out of 22 phosphorylation sites for α-casein. Moreover, the mesoporous ZrO2 and HfO2 can be reused after a simple solution regeneration procedure with comparable enrichment performance to that of fresh materials. Mesoporous ZrO2 and HfO2 nanomaterials hold great promise for applications in MS-based phosphoproteomics.
doi:10.1021/ac100877a
PMCID: PMC2936271  PMID: 20704311
19.  α-fetoprotein involvement during glucocorticoid-induced precocious maturation in rat colon 
AIM: To investigate the role of α-fetoprotein (AFP), a cancer-associated fetal glycoprotein, in glucocorticoid-induced precocious maturation in rat colon.
METHODS: Colons from suckling Sprague-Dawley rats were used in this study. Corticosterone acetate at a dose of 100 μg/g body weight was given to normal pups on days 7, 9 and 11 after birth to induce hypercorticoidism. Control animals were injected with identical volumes of normal saline. Some rats receiving corticosterone 7 d after birth were also treated with mifepristone (RU38486), a glucocorticoid cytoplasm receptor antagonist to investigate the effects of glucocorticoids (GCs). The morphological changes of the crypt depth and villous height of the villous zone in colon were observed as indices of colon maturation. Expression levels of AFP in colons were detected by reverse transcriptase polymerase chain reaction and Western blotting. To identify the cellular localization of AFP in developing rat colons, double-immunofluorescent staining was performed using antibodies to specific mesenchymal cell marker and AFP.
RESULTS: Corticosterone increased the crypt depth and villous height in the colon of 8- and 10-d-old rats with hypercorticoidism compared with that in the control animals (120% in 8-d-old rats and 118% in 10-d-old rats in villous height, P = 0.021; 145% in 8-d-old rats and 124% in 10-d-old rats in crypt depth, P = 0.017). These increases were accompanied by an increase of AFP expression in both mRNA and protein (2.5-folds in 8-d-old and 2.5-folds in 10-d-old rats higher than in control animals, P = 0.035; 1.8-folds in 8-d-old and 1.3-folds in 10-d-old rats higher than in control animals, P = 0.023). Increased crypt depth and villous height and increased expression of AFP in the colon of rats with hypercorticoidism were blocked by mifepristone. Both had positive staining for AFP or vimentin, and overlapped in mesenchymal cells at each tested colon.
CONCLUSION: GCs promote the development of rat colon. AFP appears to be involved, in part, in mediating the effects of GCs in the developmental colon.
doi:10.3748/wjg.v17.i24.2933
PMCID: PMC3129507  PMID: 21734804
Glucocorticoids; α-fetoprotein; Precocious maturation; Colon; Rat
20.  Deciphering Modifications in Swine Cardiac Troponin I by Top-Down High-Resolution Tandem Mass Spectrometry 
Cardiac troponin I (cTnI) is an important regulatory protein in cardiac muscle and its modification represents a key mechanism in the regulation of cardiac muscle contraction and relaxation. cTnI is often referred to as the “gold-standard” serum biomarker for diagnosing patients with acute cardiac injury since it is unique to the heart and released into the circulation following necrotic death of cardiac tissue. The swine (Sus scrofa) heart model is extremely valuable for cardiovascular research since the heart anatomy and coronary artery distribution of swine are almost identical to those of humans. Herein we report a comprehensive characterization of the modifications in swine cTnI using top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification. High-resolution high accuracy mass spectrometry revealed that swine cTnI affinity purified from domestic pig hearts was N-terminally acetylated and phosphorylated. Electron capture disassociation is uniquely suited for localization of labile phosphorylations, which unambiguously identified Ser22/Ser23 as the only basally phosphorylation sites that are well-known to be regulated by protein kinase A and protein kinase C. Moreover, a combination of tandem mass spectrometry with sequence homology alignment effectively localized a single amino acid polymorphism, V116A, representing a novel genetic variant of swine cTnI. Overall, our studies demonstrated the unique power of top-down high-resolution tandem mass spectrometry in the characterization of protein modifications including labile phosphorylation and unexpected sequence variants.
doi:10.1016/j.jasms.2010.02.005
PMCID: PMC3056346  PMID: 20223681
Top-Down Mass Spectrometry; Electron Capture Dissociation; Phosphorylation; Single Amino Acid Polymorphism; Cardiac Troponin I; Heart Diseases
21.  Automethylation of CARM1 allows coupling of transcription and mRNA splicing 
Nucleic Acids Research  2010;39(7):2717-2726.
Coactivator-associated arginine methyltransferase 1 (CARM1), the histone arginine methyltransferase and coactivator for many transcription factors, is subject to multiple post-translational modifications (PTMs). To unbiasedly investigate novel CARM1 PTMs we employed high-resolution top-down mass spectrometry. Surprisingly, mouse CARM1 expressed in insect and mammalian expression systems was completely dimethylated at a single site in the C-terminal domain (CTD). We demonstrate that dimethylation of CARM1 occurs both in vivo and in vitro and proceeds via an automethylation mechanism. To probe function of automethylation, we mutated arginine 551 to lysine to create an automethylation-deficient CARM1. Although mutation of CARM1's automethylation site did not affect its enzymatic activity, it did impair both CARM1-activated transcription and pre-mRNA splicing. These results strongly imply that automethylation of CARM1 provides a direct link to couple transcription and pre-mRNA splicing in a manner differing from the other steroid receptor coactivators. Furthermore, our study identifies a self-regulatory signaling mechanism from CARM1's catalytic domain to its CTD.
doi:10.1093/nar/gkq1246
PMCID: PMC3074151  PMID: 21138967
22.  Identification and Functional Characterization of Phosphorylation Sites on GTP Cyclohydrolase I 
Objective
The post-translational regulation of GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites.
Methods and Results
Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167 and threonine (T) 231 in HEK293 cells whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites [S51, S72, T85, T91, T103, S130, S167 and T231]. GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D or T130D mutants, but decreased in cells transfected with the T231D mutant, while cells transfected with the S167A or the S167E mutant had increased BH2. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity.
Conclusion
Our data suggest GCH-1 activity is regulated either positively by phosphorylation S51, S72, T85, T91, T103 and S130, or negatively at T231. Such information might be useful in designing new therapies aiming at improving BH4 bioavailability.
doi:10.1161/ATVBAHA.109.194464
PMCID: PMC2798731  PMID: 19762783
GTP Cyclohydrolase I; tetrahydrobiopterin; phosphorylation
23.  Mesoporous Zirconium Oxide Nanomaterials Effectively Enrich Phosphopeptides for Mass Spectrometry-based Phosphoproteomics 
This work represents the first use of mesoporous zirconium oxide nanomaterials for highly effective and selective enrichment of phosphorylated peptides.
doi:10.1039/b908788e
PMCID: PMC2790071  PMID: 19865665
24.  Differential modification of Cys10 alters transthyretin's effect on beta-amyloid aggregation and toxicity 
Tg2576 mice produce high levels of beta-amyloid (Aβ) and develop amyloid deposits, but lack neurofibrillary tangles and do not suffer the extensive neuronal cell loss characteristic of Alzheimer's disease. Protection from Aβ toxicity has been attributed to up-regulation of transthyretin (TTR), a normal component of plasma and cerebrospinal fluid. We compared the effect of TTR purified from human plasma (pTTR) with that produced recombinantly (rTTR) on Aβ aggregation and toxicity. pTTR slowed Aβ aggregation but failed to protect primary cortical neurons from Aβ toxicity. In contrast, rTTR accelerated aggregation, while effectively protecting neurons. This inverse correlation between Aβ aggregation kinetics and toxicity is consistent with the hypothesis that soluble intermediates rather than insoluble fibrils are the most toxic Aβ species. We carried out a detailed comparison of pTTR with rTTR to ascertain the probable cause of these different effects. No differences in secondary, tertiary or quaternary structure were detected. However, pTTR differed from rTTR in the extent and nature of modification at Cys10. We hypothesize that differential modification at Cys10 regulates TTR's effect on Aβ aggregation and toxicity.
doi:10.1093/protein/gzp025
PMCID: PMC2719498  PMID: 19549717
Alzheimer's disease; beta-amyloid; post-translational modification; transthyretin
25.  Global transcriptome profiling of wild soybean (Glycine soja) roots under NaHCO3 treatment 
BMC Plant Biology  2010;10:153.
Background
Plant roots are the primary site of perception and injury for saline-alkaline stress. The current knowledge of saline-alkaline stress transcriptome is mostly focused on saline (NaCl) stress and only limited information on alkaline (NaHCO3) stress is available.
Results
Using Affymetrix® Soybean GeneChip®, we conducted transcriptional profiling on Glycine soja roots subjected to 50 mmol/L NaHCO3 treatment. In a total of 7088 probe sets, 3307 were up-regulated and 5720 were down-regulated at various time points. The number of significantly stress regulated genes increased dramatically after 3 h stress treatment and peaked at 6 h. GO enrichment test revealed that most of the differentially expressed genes were involved in signal transduction, energy, transcription, secondary metabolism, transporter, disease and defence response. We also detected 11 microRNAs regulated by NaHCO3 stress.
Conclusions
This is the first comprehensive wild soybean root transcriptome analysis under alkaline stress. These analyses have identified an inventory of genes with altered expression regulated by alkaline stress. The data extend the current understanding of wild soybean alkali stress response by providing a set of robustly selected, differentially expressed genes for further investigation.
doi:10.1186/1471-2229-10-153
PMCID: PMC3017823  PMID: 20653984

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