Proteome analysis of abundantly expressed proteins from unfed larvae of the cattle tick, Boophilus microplus

Protein expression in unfed larvae of the cattle tick, Boophilus microplus, was characterized using gel electrophoresis and mass spectrometry in an effort to assemble a database of proteins produced at this stage of development. Soluble and insoluble proteins were extracted and resolved by two-dimensional (2D) gel electrophoresis. Twenty abundantly expressed larval proteins were selected for peptide mass mapping and for peptide sequencing by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) and quadrupole time-of-flight (Q-ToF) tandem mass spectrometry (MS), respectively. Only one protein, tropomyosin, was unequivocally identified from its peptide mass map. Ten proteins were assigned putative identities based on BLAST searching of heterologous databases with peptide sequences. These included a cytoskeletal protein (troponin I), multiple cuticular proteins, a glycine-rich salivary gland-associated protein and proteins with a presumed housekeeping role (arginine kinase, a high-mobility group protein and a small heat shock protein). Eight additional proteins were identified by searching translated open reading frames of a B. microplus EST database (unpublished): putative fatty-acid binding protein, thioredoxin, glycine-rich salivary gland protein and additional cuticular proteins. One remaining protein was not identifiable, suggesting it may be a novel molecule. The ongoing assembly of this database contributes to our understanding of proteins expressed by the tick and provides a resource that can be mined for molecules that play a role in tick-host interactions.     

The identification of allergen proteins in sugar beet (Beta vulgaris) pollen causing occupational allergy in greenhouses

Background

During production of sugar beet (Beta vulgaris) seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen.

Methods

Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry.

Results

A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences.

Conclusion

Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2), and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.     

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.     

Identification of mouse brain proteins associated with isoform 3 of metallothionein

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.     

Proteomics and immunological analysis of a novel shrimp allergen,Pen m 2

Shellfish are a common cause of adverse food reactions in hypersensitive individuals and shrimp is one of the most frequently reported causes of allergic reactions. A novel allergen from Penaeus monodon, designated Pen m 2, was identified by two-dimensional immunoblotting using sera from subjects with shrimp allergy, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptide digest. This novel allergen was then cloned and the amino acid sequence deduced from the cDNA sequence. The cloned cDNA encoded a 356-aa protein with an acetylated N terminus at Ala2, identified by postsource decay analysis. Comparison of the Pen m 2 sequence with known protein sequences revealed extensive similarity with arginine kinase (EC 2.7.3.3) from crustaceans. Pen m 2 was purified by anion exchange chromatography and shown to have arginine kinase activity and to react with serum IgE from shrimp allergic patients and induce immediate type skin reactions in sensitized patients. Using Pen m 2-specific antisera and polyclonal sera from shrimp-sensitive subjects in a competitive ELISA inhibition assay, Pen m 2 was identified as a novel cross-reactive Crustacea allergen. This novel allergen could be useful in allergy diagnosis and in the treatment of Crustacea-derived allergic disorders.     

Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials

To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t-test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed.     

A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.     

A proteomic approach to characterize protein shedding

Shedding (i.e. proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fractions, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. The resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 45 membrane-associated proteins were identified including 22 that contain at least one transmembrane domain and 23 that indirectly associate with the extracellular surface of the plasma membrane. Of the 22 transmembrane proteins, 18 were identified by extracellular peptides providing strong evidence they originate from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified two proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78 kDa glucose-regulated protein and lactate dehydrogenase A. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78 kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that tandem mass spectrometry can be used to identify shed proteins and to estimate changes in protein abundance.     

One-dimensional proteomic mapping of human liver cytochromes P450

A method for constructing one-dimensional proteomic maps (1D-PM) based on mass spectrometric identification of proteins from adjacent slices of one-dimensional electrophoregram has been developed. For the proteomic mapping, gel lanes were sectioned into slices less than 0.2 mm thick and each slice was subjected to enzymatic hydrolysis. The resultant mixture of peptide fragments was analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteins were identified by the mass spectra obtained. Data on peptide fragments and corresponding identified proteins were presented as a 1D-PM. Proteomic maps were constructed by assigning individual proteins to gel slices based on number of matching peptides in a corresponding MS-data. On 1D-PM of human liver microsomal fraction, 18 proteins were identified in the region of 40–65 kDa. These included 12 membrane proteins belonging to the superfamily of cytochromes P450. Pooling of mass spectrometric data, obtained from several adjacent gel slices (molecular zooming) increased sequence coverage of CYP2A (cytochrome P450 family 2A). The maximal coverage of 66% significantly exceeded the level of 48% that could be obtained using one (even the most informative) slice. This method can be applied to the proteomic profiling of membrane-bound proteins.     

Proteomic analysis of the mosquito Aedes aegypti midgut brush border membrane vesicles

We analyzed brush border membrane vesicle proteins from isolated midguts of the mosquito Aedes aegypti, by two proteomic methods: two-dimensional gel electrophoresis (isoelectric focusing and SDS-PAGE) and a shotgun two-dimensional liquid chromatographic (LS/LS) approach based on multidimensional protein identification technology (MudPIT). We were interested in the most abundant proteins of the apical brush border midgut membrane. About 400 spots were detected on 2D gels and 39 spots were cored and identified by mass spectrometry. 86 proteins were identified by MudPIT. Three proteins, arginine kinase, putative allergen and actin are shown to be the most predominant proteins in the sample. The total number of 36 proteins detected by both methods represents the most abundant proteins in the BBMV.     

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.     

Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods

We compared detection sensitivity and protein sequence coverage of the adenovirus type 5 proteome achievable by liquid chromatography and tandem mass spectroscopy (LC/MS/MS) using three sample preparation and clean up methods. Tryptic digestion was performed on either purified viral proteins or whole virus, and followed by shotgun sequencing using tandem mass spectrometry for peptide identification. We used a recombinant adenovirus type 5 as a test system. The methods included separation of adenoviral proteins by reversed-phase high-performance liquid chromatography followed by tryptic digestion and analysis by LC/MS/MS. Alternatively, the purified whole virus was digested with trypsin and the peptides separated either by one-dimensional (reversed-phase) or by two-dimensional (cation exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. A total of 11 protein species were identified from 154 peptides. All of the major viral proteins were found. In addition, two minor proteins, the 23 kDa viral protease and the late L1 protein, were identified for the first time by chromatography based assays. The 23 kDa viral protease, present at only 10 copies per virus, and representing 0.2% of the protein content of the virus, was detected by the 2D LC/MS/MS analysis of the whole virus digest from a sample containing only 70 fmols of the protein. This demonstrates the high sensitivity and selectivity of the method. The 2D LC/MS/MS analysis of the whole virus digest was also able to detect all viral proteins with copy numbers at or above 10/virus particle, with broad coverage of the amino acid sequences. Coverage ranged from 2 to 54%, a majority between 20 and 35%, suggesting the possibility of using this analysis to assess the purity of the virus preparations. This broad coverage may also provide a useful approach to identify posttranslational modifications on the structural proteins of the adenovirus.     

Site recognition and substrate screens for PKN family proteins

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR (epidermal growth factor receptor), was identified. This identified Thr??? in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr??? is shown to be modulated by PKN in vivo.     

Characterization of a bovine pregnancy-associated protein using two-dimensional gel electrophoresis, N-terminal sequencing and mass spectrometry

Bovine pregnancy-associated protein (bPAP) isolated from pregnant bovine urines by two-dimensional electrophoresis (2-DE) was characterized by N-terminal sequencing, internal sequencing, and mass spectrometric analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry. Database search using the amino acid sequences and the peptide mass profiles showed that the protein is a novel bovine pregnancy-associated protein of which the N-terminus has a high similarity to collagen alpha. The protein has a molecular mass of 21 kDa and a pI of 6.1. The expression profiles of the protein from the urine of 30 pregnant and 20 nonpregnant cows characterized by 2-DE indicated that the expression of bPAP during pregnancy increased to over nmol from the pmol level basal expression of bPAP at the nonpregnant state with < 3% of false negatives and < 10% of false positives. Using the peptide sequence information, polyclonal antibodies against the bPAP protein were generated. The purified polyclonal antibodies against the peptide sequences of bPAP detected the 21 kDa protein on the blots of pregnant cow urine by Western blot analysis. In addition, analysis showed that the expression of bPAP in the urine is associated with pregnancy, but that the urine concentration of bPAP is not correlated with the duration of the pregnancy.     

The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare,primitive hematopoietic cells from patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Stephen D. Griffiths and John Burthem contributed equally to this publication. This work is supported by The Leukaemia Research Fund (UK).     

Skin mucus proteins of lumpsucker (Cyclopterus lumpus)

Fish skin mucus serves as a first line of defense against pathogens and external stressors. In this study the proteomic profile of lumpsucker skin mucus was characterized using 2D gels coupled with tandem mass spectrometry. Mucosal proteins were identified by homology searches across the databases SwissProt, NCBInr and vertebrate EST. The identified proteins were clustered into ten groups based on their gene ontology biological process in PANTHER (www.patherdb.org). Calmodulin, cystatin-B, histone H2B, peroxiredoxin1, apolipoprotein A1, natterin-2, 14-3-3 protein, alfa enolase, pentraxin, warm temperature acclimation 65 kDa (WAP65kDa) and heat shock proteins were identified. Several of the proteins are known to be involved in immune and/or stress responses. Proteomic profile established in this study could be a benchmark for differential proteomics studies.     

Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex

The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task. Here we describe and apply a comprehensive method that combines a mild affinity purification procedure with a multiplexed mass spectrometry approach for the in-depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels. The exosome is an ensemble of primarily 3' --> 5' exoribonucleases and plays a major role in RNA metabolism. The complex has been reported to consist of 11 proteins in molecular mass ranging from 20 to 120 kDa. By using native macromolecular mass spectrometry we measured accurate masses (around 400 kDa) of several (sub)exosome complexes. Combination of these data with proteolytic peptide LC tandem mass spectrometry using a linear ion trap coupled to a FT-ICR mass spectrometer and intact protein LC mass spectrometry provided us with the identity of the different exosome components and (sub)complexes, including the subunit stoichiometry. We hypothesize that the observed complexes provide information about strongly and weakly interacting exosome-associated proteins. In our analysis we also identified for the first time phosphorylation sites in seven different exosome subunits. The phosphorylation site in the Rrp4 subunit is fully conserved in the human homologue of Rrp4, which is the only previously reported phosphorylation site in any of the human exosome proteins. The described multiplexed mass spectrometry-based procedure is generic and thus applicable to many different types of cellular molecular machineries even if they are expressed at endogenous levels.     

Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803

Proteome analysis by gel-free "shotgun" proteomics relies on the simplification of a peptide mixture before it is analyzed in a mass spectrometer. While separation on a reverse-phase (RP) liquid chromatographic column is widely employed, a variety of other methods have been used to fractionate both proteins and peptides before this step. We compared six different protein and peptide fractionation workflows, using Synechocystis sp. PCC 6803, a useful model cyanobacterium for potential exploitation to improve its production of hydrogen and other secondary metabolites. Pre-digestion protein separation was performed by strip-based isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis, or weak anion exchange chromatography, while pre-RP peptide separation was accomplished by isoelectric focusing (IEF) or strong cation exchange chromatography. Peptides were identified using electrospray ionization quadrupole time of flight-tandem mass spectrometry. Mass spectrometry (MS) and tandem mass spectra were analyzed using ProID software employing both a single organism database and the entire NCBI non-redundant database, and a total of 776 proteins were identified using a stringent set of selection criteria. Method comparisons were made on the basis of the results obtained (number and types of proteins identified), as well as ease of use and other practical aspects. IEF-IEF protein and peptide fractionation prior to RP gave the best overall performance.