Proteome response to the panneural expression of human wild-type alpha-synuclein: a Drosophila model of Parkinson's disease

The alpha-synuclein protein is associated with several neurodegenarative diseases, including Parkinson's disease (PD). In humans, only mutated forms of alpha-synuclein are linked to PD; however, panneural expression of human wild-type (WT) alpha-synuclein induces Parkinson's like-symptoms in Drosophila. Here, we report a quantitative proteomic analysis of WT alpha-synuclein transgenic flies with age-matched controls at the presymptomatic stage utilizing a global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry. The analysis includes two biological replicates, in which samples are isotopically labeled in forward and reverse directions. In total, 229 proteins were quantified from assignments of at least two peptide sequences. Of these, 188 (82%) proteins were detected in both forward and reverse labeling measurements. Twelve proteins were found to be differentially expressed in response to the expression of human WT alpha-synuclein; down-regulations of larval serum protein 2 and fat body protein 1 levels were confirmed by Western blot analysis. Gene Ontology analysis indicates that the dysregulated proteins are primarily associated with cellular metabolism and signaling, suggesting potential contributions of perturbed metabolic and signaling pathways to PD. An increased level of the iron (III)-binding protein, ferritin, typically found in the brains of PD patients, is also observed in presymptomatic WT alpha-synuclein expressing animals. The observed alterations in both pathology-associated and novel proteins may shed light on the pathological roles of alpha-synuclein that may lead to the development of diagnostic strategies at the presymptomatic stage.     

Effect of CDP-choline on the biosynthesis of nucleic acids and proteins in brain regions during hypoxia

The effect of CDP-choline on the in vivo incorporation of labeled precursors into DNA, RNA, and proteins in cerebral hemispheres, cerebellum, and brainstem of guinea pigs after hypoxic treatment was studied. The labeling of macromolecules extracted from the various subcellular fractions of these brain regions was also determined. Hypoxic treatment affected macromolecular labeling to a different extent in the three brain regions examined. CDP-choline treatment was not able to reverse the effect of hypoxia on DNA labeling, but it was able to remove the effect of hypoxia on RNA and protein labeling. The action of CDP-choline was particularly evident on the labeling of RNA in nuclei and mitochondria of the cerebellum and on the labeling of proteins in microsomes of the three brain regions examined.     

Beta-lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana

New activity-based probes are essential for expanding studies on the hundreds of serine and cysteine proteases encoded by the genome of Arabidopsis thaliana. To monitor protease activities in plant extracts, we generated biotinylated peptides containing a beta-lactone reactive group. These probes cause strong labeling in leaf proteomes. Unexpectedly, labeling was detected at the N terminus of PsbP, nonproteolytic protein of photosystem II. Inhibitor studies and reverse genetics led to the discovery that this unusual modification is mediated by a single plant-specific, papain-like protease called RD21. In cellular extracts, RD21 accepts both beta-lactone probes and peptides as donor molecules and ligates them, probably through a thioester intermediate, to unmodified N termini of acceptor proteins.     

Two classes of phosphotyrosine-containing proteins detected by labeling with 32P and 125I in HeLa cells

There are two classes of proteins that can be phosphorylated on tyrosine in HeLa cells. One class can be detected by metabolic labeling with [32P]Pi and affinity chromatography using anti-phosphotyrosine antibodies. The other cannot be detected by this technique but can be detected among the proteins which bind to the antibodies by in vitro iodination with 125I. Presumably proteins of the second class contain phosphotyrosine at which the phosphate undergoes very slow turnover. The incubation of cells in phosphate-minus medium caused a marked reduction in the levels of phosphotyrosine-containing proteins, this explaining the failure of detection of the second class proteins even after prolonged labeling with [32P]Pi.     

A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self-incompatible species Solanum chacoense Bitt

We have used 2-DE for a time-course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein-specific staining. Using a pI range of 4-7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro-Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC-MS/MS using a hybrid quadrupole-TOF (Q-TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.     

Quantitative proteomics of a presymptomatic A53T alpha-synuclein Drosophila model of Parkinson disease

A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was used for quantitative proteome analysis of a presymptomatic A53T alpha-synuclein Drosophila model of Parkinson disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 ( approximately 71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T alpha-synuclein Drosophila; up-regulation of troponin T and down-regulation of fat body protein 1 were confirmed by Western blot analysis. Elevated expressions of heat shock protein 70 cognate 3 and ATP synthase are known to be directly involved in A53T alpha-synuclein-mediated toxicity and PD; three up-regulated proteins (muscle LIM protein at 60A, manganese-superoxide dismutase, and troponin T) and two down-regulated proteins (chaoptin and retinal degeneration A) have literature-supported associations with cellular malfunctions. That these variations were observed in presymptomatic animals may shed light on the etiology of PD. Protein interaction network analysis indicated that seven proteins belong to a single network, which may provide insight into molecular pathways underlying PD. Gene Ontology analysis indicated that the dysregulated proteins are primarily associated with membrane, endoplasmic reticulum, actin cytoskeleton, mitochondria, and ribosome. These associations support prior findings in studies of the A30P alpha-synuclein Drosophila model (Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Protein expression in a Drosophila model of Parkinson's disease. J. Proteome Res. 6, 348-357; Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Lifetime proteomic profiling of an A30P alpha-synuclein Drosophila model of Parkinson's disease. J. Proteome Res. 6, 3729-3738) that defects in cellular components such as actin cytoskeleton and mitochondria may contribute to the development of later symptoms.     

Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus

Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of alpha-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.     

p-Diazobenzoyl biocytin--a new biotinylating reagent for the labeling of tyrosines and histidines in proteins

A new biotin-containing reagent, p-diazobenzoyl biocytin (DBB), has been developed for labeling tyrosines and histidines in proteins. The reagent has used to label these residues in both model proteins and erythrocyte membrane proteins on dot blots and blot transfers. In some cases, sub-nanogram levels of individual proteins could be detected. The utility of DBB as a versatile alternative to biotin-containing N-hydroxysuccinimide esters for the general labeling of proteins is discussed.     

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.     

Immunochemical detection of adenine nucleotide-binding proteins with antibodies to 5'-p-fluorosulfonylbenzoyladenosine

5'-p-Fluorosulfonylbenzoyladenosine (FSBA) is a useful reagent for the affinity labeling of adenine nucleotide binding proteins. We have developed an immunochemical approach to the detection of proteins that have been covalently modified with FSBA, which provides an alternative to the use of a radiolabeled ligand. Antibodies have been prepared against FSBA-modified glutamate dehydrogenase and purified by chromatography on ATP-agarose. The resulting affinity-purified antibodies react on Western blots only with proteins that have been labeled previously with the affinity reagent. The degree of immunoreactivity on Western blots correlates well with the extent of covalent modification as shown by studies on the modification and inhibition of the catalytic subunit of cAMP-dependent protein kinase. In crude cellular extracts, numerous proteins can be labeled with FSBA and then detected by using this approach. The labeling and subsequent detection of these proteins can be blocked by including an excess of MgATP, which competes with FSBA for nucleotide-binding sites. The labeling of specific proteins in crude mixtures is saturable, as shown by labeling studies of p56lck, a protein-tyrosine kinase that is abundantly expressed in membranes from the T lymphoma cell line LSTRA.     

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.     

Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.     

Performance of isobaric and isotopic labeling in quantitative plant proteomics

Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins.     

Exchange and stability of HeLa ribosomal proteins in vivo.

The relative stabilities of individual HeLa ribosomal proteins and their capacity for exchange between ribosome-bound and -free states in the cytoplasm were examined. Most ribosomal proteins on cytoplasmic ribosomes were found to have uniform, high stability as measured by comparing the short term (12-hour) to steady state (3-day) labeling ratios determined for each ribosomal protein. This would be expected if the proteins in ribosomes either were all stable or were all degraded as a unit. The data do not rule out the possibility that individual proteins have different stabilities prior to their assembly into ribosomes. Four proteins labeled atypically. One large subunit protein (L5) had a lower than average ratio. We interpret this low ratio as being due to a large free pool of this protein. Three proteins (L10, L28, S2) had higher than average ratios, interpreted as being due to reduced protein stability. Two of these proteins (L10, L28) with high ratios were also found to exchange in vivo. The exchangeable proteins may be subject to increased degradation during the time that they spend in the exchangeable free pool. The third protein (S2) with an atypically high ratio is thought to be degraded or altered while on the ribosome, or slowly lost as ribosomes age, because exchange of this protein was not detected. These interpretations and some alternate interpretations are explained. The exchange of three large subunit proteins (L10, L19, L28) was detected by labeling of protein after ribosome synthesis had been inhibited with actinomycin D. Autoradiography of two-dimensional polyacrylamide gels showed labeling of these spots.     

Characterization of caspase proteases in cytokine-dependent myeloid progenitor cells using enzyme affinity labeling

Bone marrow-derived myeloid progenitor cells are dependent on the presence of cytokines such as interleukin-3 (IL-3) for their survival. The withdrawal of IL-3 from IL-3-dependent myeloid progenitors results in death via an apoptotic program. Previous studies have shown that IL-3 withdrawal induces the activities of caspase proteases. However, the molecular identities of myeloid progenitor caspases have not been determined. In this study, we used an affinity labeling reagent (biotin-YVAD-acyloxymethyl ketone) that binds to processed active caspase subunits, to study caspase activation in 32D and FDCP-1 myeloid progenitor cells. After IL-3 withdrawal, we detected affinity labeling of caspase subunits of 20, 17, and 16 kDa in both cell lines. Surprisingly, affinity labeling of the 20- and 17-kDa proteins, but not the 16-kDa protein, was also detected in healthy cells maintained in the presence of IL-3. By contrast, in cytokine-independent cell lines, affinity labeling of caspase subunits was detected only after treatment with an apoptotic stimulus. Immunoblotting experiments showed that caspase-3 constitutes at least a portion of the 20- and 17-kDa affinity-labeled proteins detected in the myeloid progenitor cell lines. Taken together, these data provide direct evidence of caspase activation in cytokine-dependent myeloid progenitors, and suggest that unique apoptotic pathways may exist in these cells.     

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis

Blue native gel electrophoresis (BN–PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN–PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN–PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed.     

Newly synthesized proteins in seminiferous intertubular and intratubular compartments of the rat testis

Two-dimensional gel electrophoresis combined with autoradiography and Western blot procedures have been used to characterize newly synthesized proteins in testicular intertubular fluid (TIF) and seminiferous tubular fluid (SNF). Fluids were collected following in vivo and in vitro intratesticular injection of [35S]methionine into control and hypophysectomized adult rats. A discrete number of [35S]methionine-labeled proteins were detected within TIF and SNF. Their presence and relative abundance varied according to in vivo and in vitro labeling conditions. While two major blood plasma proteins, albumin and transferrin, were radioactively labeled after in vivo labeling, these two proteins were insignificantly labeled in samples collected after in vitro labeling. Three acidic proteins, possibly secreted by Sertoli cells (Mr = 72,000, 45,000 and 35,000), were more abundant in TIF samples collected after in vitro [35S]methionine labeling than after in vivo labeling. Incubated seminiferous tubules and TIF of hypophysectomized rats showed a decrease in [35S]methionine-labeling intensity of the Mr = 72,000 acidic protein, possibly reflecting changes in the seminiferous epithelium caused by pituitary hormonal deprivation. Autoradiographs of TIF and most remarkably, of SNF, showed many protein spots that suggested cell breakage and leakage during sample collection. Results of this study suggest that most albumin and transferrin found in TIF and SNF have an extratesticular origin and that proteins secreted by the Sertoli cell can gain access to both TIF and SNF.