The cystic fibrosis transmembrane conductance regulator (CFI'R) gene encodes an apical membrane CI-channel regulated by protein phosphorylation. To identify CAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe") nitrilotriacetic (NTA) Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBDl -R protein from CFTR. Fe3+-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HP03 and HIPOl from the parent ions. Peptide sequence and phosphorylation at CFTR residues 66"Ser, 737Ser, and 79sSer were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 7ooSer, '"Ser, 768Ser, and "'Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 7s3Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 7s3Ser in full-length CFTR. Keywords: CFTR; mass spectrometry; phosphorylationThe cystic fibrosis transmembrane conductance regulator gene encodes an apical membrane C1-channel (Riordan et al., 1989). Expression of CFTR results in increased C1-conductance following

To study interactions between the contiguous NBD1 and R domains of CFTR, wild-type and DeltaF508 NBD1-R (amino acids 404-830, in fusion with His6 tag) were expressed as single proteins in Escherichia coli. NBD1-R (10-25 mg/L culture) was purified from inclusion bodies in 8 M urea by Ni-affinity chromatography, and renatured by rapid dilution at pH 5. In vitro phosphorylation by protein kinase A increased the apparent size of NBD1-R from approximately 52 to approximately 56 kDa by SDS-PAGE. The fluorescent ATP analogue TNP-ATP bound to renatured NBD1-R with of 0.81 +/- 0.1 microM (wild-type), 0.93 +/- 0.1 microM (wild-type, phosphorylated), 0.75 +/- 0.1 microM (DeltaF508 NBD1-R), and 0.72 +/- 0.1 microM (DeltaF508 NBD1-R, phosphorylated) with a stoichiometry of approximately 1 TNP-ATP site per NBD1-R molecule; TNP-ATP binding was reversed by ATP, AMP-PCP, and AMP-PNP with KIs of approximately 3.2, 4.2, and 4.6 mM, respectively. Secondary structure analysis by circular dichroism gave 19% alpha-helix, 43% beta-sheet and turn, and 38% "other" structure. To determine if nucleotide binding to NBD1 influenced R domain phosphorylation, NBD1-R was in vitro phosphorylated with protein kinase A and [gamma-32P]ATP in the presence of AMP-PCP, AMP-PNP, or TNP-ATP. Whereas the nucleotide analogues did not affect 32P-incorporation in control proteins (Kemptide, GST-R domain), phosphorylation of NBD1-R was reduced >75% by AMP-PNP or AMP-PCP (0.25 mM) and >50% by TNP-ATP (0.25 microM). Analysis of phosphorylation sites indicated that inhibition involved multiple sites in NBD1-R, including serines 660, 712, 737, 795, and 813. These results establish the conditions for NBD1-R expression, purification, and renaturation. The inhibition of R domain phosphorylation by nucleotide binding to the NBD1 domain indicates significant domain-domain interactions and suggests a novel mechanism for regulation of CFTR phosphorylation.

Inner ear dysfunction is often associated with defective hair cells. Therefore, hair cells are the focus of study in many of the mouse mutants showing auditory and vestibular deficits. However, harvesting sufficient numbers of hair cells from the tiny bony mouse inner ear for proteomic analysis is challenging. New approaches that would take advantage of mouse mutants and avoid processing steps, such as decalcification or microdissetion, would be more suitable for proteomic analysis. Here, we propose a novel approach called SSUMM-Subtractive Strategy Using Mouse Mutants. SSUMM takes advantage of the differences between control and affected or mutant samples. We predict that SSUMM would be a useful method in proteomics, especially in those cases in which the investigator must work with small numbers of diverse cell types from a tiny organ. Here, we discuss the potential utility of SSUMM to unravel the protein expression profiles of hair cells using the Pou4f3 mouse mutant as an example. Pou4f3 mutant mice exhibit a total loss of inner and outer hair cells, but supporting cells remain relatively intact in the cochlea, thus providing an excellent model for identifying proteins and transcripts that are specific to the hair cell at all life stages. SSUMM would maximize the sensitivity of the analyses while obviating the need for tedious sessions of microdissection and collection of hair cells. By comparing the mutant to control ears at specific time points, it is possible to identify direct targets of a gene product of interest. Further, SSUMM could be used to identify and analyze inner ear development markers and other known genes/proteins that are coexpressed in the ear. In this short technical report, we also discuss protein-profiling approaches suitable for SSUMM and briefly discuss other approaches used in the field of proteomics.☆ General Electric Company reserves the right, subject to regulatory approval if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Representative for the most current information.

Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxidonitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage.cigarette smoke | emphysema | vitamin C | ascorbate | Rtp801

The use of two-dimensional gel electrophoresis for differential analysis in proteomics was revolutionized by the introduction of 2-D fluorescence difference gel electrophoresis (2-D DIGE). This fluorescence-based technique allows the use of multiplexed samples and an internal standard that virtually eliminates gel-to-gel variability, resulting in increased confidence that differences found between samples are due to real induced changes, rather than inherent biological variation or experimental variability. 2-D DIGE has quickly become the "gold standard" for gel-based proteomics for studying tissues, as well as cell culture and bodily fluids such as serum or plasma. This chapter will describe the basic 2-D DIGE technique using minimal labeling, image acquisition using high-quality fluorescence scanners, and software capable of analyzing the multiplexed images and normalizing the data using the internal standard.

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The reproductive performance of a herd is the biggest factor affecting production and product quality of livestock. Thus, a decline of male fertility represents a dramatic economic loss in beef and dairy industries. Caused by molecular defects in the spermatozoa, uncompensatory infertility is a current challenge for the cattle industry, because even with normal sperm morphology, motility, and number, fertility of bulls is still sub-par. Thus, the objective of this study was to determine the global proteome of spermatozoa collected from bulls with different fertility and study the proteins playing a role in uncompensatory infertility. We performed difference gel electrophoresis (2D-DIGE) using cryopreserved sperm from a total of 6 bulls. Spermatozoa were thawed, purified by percoll gradient, and washed in PBS solution. For each bull, a pellet of 100 million sperm cells was resuspended in the 2D-DIGE labeling buffer, and the total protein was quantified. Cell lysates were separately labeled with CyDye DIGE Fluor dyes (and reciprocally with different dyes) and multiplexed in pairs on 3 gels. The samples were focused according to their isoelectric point through an Immobiline DryStrip, pH4-7, using a IPGphor 2, followed by separation on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (21.5–200 kDa) using a DALTsix. The resulting gels were scanned, and their topographic digital maps were used for algorithmic spot matching, background normalization, spot differences quantification, and elimination of artifacts (DeCyder 2-D Differential Analysis Software, GE Healthcare Life Sciences, Piscataway, NJ, USA). The results showed between 2600 and 2800 proteins with high confidence. Compared to the high-fertile bulls, 30 proteins were increased, and 27 were decreased in the low-fertility bulls within a 2-fold range. The largest significant increase and decrease were 3.97- and 2.4-fold, respectively. The identification of these differentially represented proteins is in progress. However, our results provided a panoramic view of sperm proteome from bulls of different fertility and thus paved the way for research on mechanisms of uncompensatory bull infertility.