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author:("Hao, zhizi")
1.  Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution* 
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
PMCID: PMC2849712  PMID: 20037178
2.  On-Line LC-MS Approach Combining Collision-Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), and CID of an Isolated Charge-Reduced Species for the Trace-Level Characterization of Proteins with Post-Translational Modifications 
Journal of proteome research  2007;6(11):4230-4244.
We have expanded our recent on-line LC-MS platform for large peptide analysis to combine CID, electron transfer dissociation (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to determine sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e. 2,000 to 10,000 Da) from complex proteins, such as β-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low fmol level. The incorporation of an additional CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (∼ >1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near continuous product ion series. The identification of N-linked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymatic peptide mixtures over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated near continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g.≤ 2+ ions), which lack extensive ETD or CRCID fragmentation, can be characterized by the CID-MS2 step. A more targeted approach can then be followed in subsequent LC-MS runs to obtain additional information, if needed. Overall, the recently introduced ETD provides not only useful structural information but also enhances the confidence of all assignments. The sensitivity of this new approach on the chromatographic time scale is similar to the previous Extended Range Proteomic Analysis (ERPA) using CID-MS2 and CID-MS3. The new LC-MS platform can be anticipated to be a useful approach for the comprehensive characterization of complex proteins.
PMCID: PMC2557440  PMID: 17900180
3.  Cloning, Expression, and Characterization of Cadmium and Manganese Uptake Genes from Lactobacillus plantarum 
Applied and Environmental Microbiology  1999;65(11):4746-4752.
An Mn2+ and Cd2+ uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd2+ sensitivity as well as energy-dependent Cd2+ uptake activity. Both transcription and translation of mntA were induced by Mn2+ starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd2+ uptake systems have been identified in L. plantarum: one is a high-affinity Mn2+ and Cd2+ uptake system that is expressed in Mn2+-starved cells, and the other is a nonsaturable Cd2+ uptake system that is expressed in Cd2+-sufficient cells (Z. Hao, H. R. Reiske, and D. B. Wilson, Appl. Environ. Microbiol. 65:592–99, 1999). MntA was not detected in an Mn2+-dependent mutant of L. plantarum which had lost high-affinity Mn2+ and Cd2+ uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn2+ and Cd2+ transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn2+- and Cd2+-binding sites of MntA are discussed. A second clone containing a low-affinity Cd2+ transport system was also isolated.
PMCID: PMC91639  PMID: 10543781
4.  Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants 
Applied and Environmental Microbiology  1999;65(11):4741-4745.
Two different Cd2+ uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn2+ uptake system which also takes up Cd2+ and is induced by Mn2+ starvation. The calculated Km and Vmax are 0.26 μM and 3.6 μmol g of dry cell−1 min−1, respectively. Unlike Mn2+ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd2+ uptake is weakly inhibited by citrate. Cd2+ and Mn2+ are competitive inhibitors of each other, and the affinity of the system for Cd2+ is higher than that for Mn2+. The other Cd2+ uptake system is expressed in Mn2+-sufficient cells, and no Km can be calculated for it because uptake is nonsaturable. Mn2+ does not compete for transport through this system, nor does any other tested cation, i.e., Zn2+, Cu2+, Co2+, Mg2+, Ca2+, Fe2+, or Ni2+. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn2+-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn2+ for growth as the parental strain. Mn2+ starvation-induced Cd2+ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn2+ or Cd2+ accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn2+ and Cd2+ uptake system.
PMCID: PMC91638  PMID: 10543780

Results 1-4 (4)