Immunogenicity and contraceptive efficacy of plant-produced putative mouse-specific contraceptive peptides.

Rodent population control through contraception requires species-specific oral contraceptive vaccines. Therefore, in this study, we produced putative mouse-specific contraceptive peptides, mZP2 (from oocyte) and mIzumo1 (from sperm), in plants using Agrobacterium-mediated transient expression. Peptides were produced separately in Nicotiana benthamiana using constructs encoding antigens containing three copies of each peptide. We also determined the immunogenicity and contraceptive effects of the plant-produced antigens in female BALB/c mice. Mice immunized subcutaneously with a relatively low amount of antigen (5 µg/dose of each peptide in a mixture) showed systemic immune responses against mZP2-3 and mIzumo1-3 antigens. Moreover, the mean litter size of mice treated with the plant-produced antigens was reduced by 39% compared to that of the control mice. Notably, there was a significant negative correlation between the number of pups born and individual antibody levels against both antigens. Immunofluorescence assays demonstrated the binding of induced antibodies to the oocytes of BALB/c and wild-type mice in vivo and in vitro, respectively. Our study demonstrate the feasibility of producing small contraceptive peptides in plants that can be further used to develop oral contraceptive vaccines against mouse populations.

Memory-Enhancing Effects of Origanum majorana Essential Oil in an Alzheimer's Amyloid beta1-42 Rat Model: A Molecular and Behavioral Study.

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used in traditional medicine since ancient times as culinary herbs and remedies. The aim of the present study was to evaluate the chemical composition, as well as the biochemical and cellular activities of freshly prepared Origanum majorana L. essential oil (OmEO) in an Alzheimer's disease (AD) amyloid beta1-42 (Aß1-42) rat model. OmEO (1% and 3%) was inhaled for 21 consecutive days, while Aß1-42 was administered intracerebroventricularly to induce AD-like symptoms. Our data demonstrate that OmEO increased antioxidant activity and enhanced brain-derived neurotrophic factor (BDNF) expression, which in concert contributed to the improvement of cognitive function of animals. Moreover, OmEO presented beneficial effects on memory performance in Y-maze and radial arm-maze tests in the Aß1-42 rat AD model.

Kininogen supports inflammation and bacterial spreading during Streptococccus Pyogenes Sepsis.

BACKGROUND: High-molecular-weight kininogen is a cofactor of the human contact system, an inflammatory response mechanism that is activated during sepsis. It has been shown that high-molecular-weight kininogen contributes to endotoxemia, but is not critical for local host defense during pneumonia by Gram-negative bacteria. However, some important pathogens, such as Streptococcus pyogenes, can cleave kininogen by contact system activation. Whether kininogen causally affects antibacterial host defense in S. pyogenes infection, remains unknown. METHODS: Kininogen concentration was determined in course plasma samples from septic patients. mRNA expression and degradation of kininogen was determined in liver or plasma of septic mice. Kininogen was depleted in mice by treatment with selective kininogen directed antisense oligonucleotides (ASOs) or a scrambled control ASO for 3 weeks prior to infection. 24 h after infection, infection parameters were determined. FINDINGS: Data from human and mice samples indicate that kininogen is a positive acute phase protein. Lower kininogen concentration in plasma correlate with a higher APACHE II score in septic patients. We show that ASO-mediated depletion of kininogen in mice indeed restrains streptococcal spreading, reduces levels of proinflammatory cytokines such as IL-1ß and IFNγ, but increased intravascular tissue factor and fibrin deposition in kidneys of septic animals. INTERPRETATION: Mechanistically, kininogen depletion results in reduced plasma kallikrein levels and, during sepsis, in increased intravascular tissue factor that may reinforce immunothrombosis, and thus reduce streptococcal spreading. These novel findings point to an anticoagulant and profibrinolytic role of kininogens during streptococcal sepsis. FUNDING: Full details are provided in the Acknowledgements section.

Drug Repositioning Inferred from E2F1-Coregulator Interactions Studies for the Prevention and Treatment of Metastatic Cancers.

Metastasis management remains a long-standing challenge. High abundance of E2F1 triggers tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a network of prometastatic transcriptional targets. Methods: To identify E2F1-coregulators, we integrated high-throughput Co-immunoprecipitation (IP)/mass spectometry, GST-pull-down assays, and structure modeling. Potential inhibitors of PPI discovered were found by bioinformatics-based pharmacophore modeling, and transcriptome profiling was conducted to screen for coregulated downstream targets. Expression and target gene regulation was validated using qRT-PCR, immunoblotting, chromatin IP, and luciferase assays. Finally, the impact of the E2F1-coregulator complex and its inhibiting drug on metastasis was investigated in vitro in different cancer entities and two mouse metastasis models. Results: We unveiled that E2F1 forms coactivator complexes with metastasis-associated protein 1 (MTA1) which, in turn, is directly upregulated by E2F1. The E2F1:MTA1 complex potentiates hyaluronan synthase 2 (HAS2) expression, increases hyaluronan production and promotes cell motility. Disruption of this prometastatic E2F1:MTA1 interaction reduces hyaluronan synthesis and infiltration of tumor-associated macrophages in the tumor microenvironment, thereby suppressing metastasis. We further demonstrate that E2F1:MTA1 assembly is abrogated by small-molecule, FDA-approved drugs. Treatment of E2F1/MTA1-positive, highly aggressive, circulating melanoma cells and orthotopic pancreatic tumors with argatroban prevents metastasis and cancer relapses in vivo through perturbation of the E2F1:MTA1/HAS2 axis. Conclusion: Our results propose argatroban as an innovative, E2F-coregulator-based, antimetastatic drug. Cancer patients with the infaust E2F1/MTA1/HAS2 signature will likely benefit from drug repositioning.

Proteome analysis reveals that de novo regenerated mucosa over fibula flap-reconstructed mandibles resembles mature keratinized oral mucosa.

AIM: The aim of this study was to determine whether intra-oral de novo regenerated mucosa (D) that grew over free fibula flap reconstructed-mandibles resembled the donor tissue i.e. external skin (S) of the lateral leg, or the recipient site tissue, i.e. keratinized oral mucosa (K). MATERIALS AND METHODS: Differential proteome analysis was performed with ten tissue samples from each of the three groups: de novo regenerated mucosa (D), external skin (S), and keratinized oral mucosa (K). Expression differences of cornulin and involucrin were validated by Western blot analysis and their spatial distributions in the respective tissues were ascertained by immunohistochemistry. RESULTS: From all three investigated tissue types a total of 1188 proteins were identified, 930 of which were reproducibly and robustly quantified by proteome analysis. The best differentiating proteins were assembled in an oral mucosa proteome signature that encompasses 56 differentially expressed proteins. Principal component analysis of both, the 930 quantifiable proteins and the 56 oral mucosa signature proteins revealed that the de novo regenerated mucosa resembles keratinized oral mucosa much closer than extra-oral skin. Differentially expressed cornification-related proteins comprise proteins from all subclasses of the cornified cell envelope. Prominently expressed in intra-oral mucosa tissues were (i) cornifin-A, cornifin-B, SPRR3, and involucrin from the cornified-cell-envelope precursor group, (ii) S100A9, S100A8 and S100A2 from the S100 group, and (iii) cornulin which belongs to the fused-gene-protein group. CONCLUSION: According to its proteome signature de novo regenerated mucosa over the free fibula flap not only presents a passive structural surface layer but has adopted active tissue function.

Deletion of the L-Lactate Dehydrogenase Gene ldh in Streptococcus pyogenes Leads to a Loss of SpeB Activity and a Hypovirulent Phenotype.

Streptococcus pyogenes uses lactic acid fermentation for the generation of ATP. Here, we analyzed the impact of a deletion of the L-lactate dehydrogenase gene ldh on the virulence of S. pyogenes M49. While the ldh deletion does not cause a general growth deficiency in laboratory media, the growth in human blood and plasma is significantly hampered. The ldh deletion strain is furthermore less virulent in a Galleria mellonella infection model. We show that the ldh deletion leads to a decrease in the activity of the cysteine protease SpeB, an important secreted virulence factor of S. pyogenes. The reduced SpeB activity is caused by a hampered autocatalytic activation of the SpeB zymogen into the mature SpeB. The missing SpeB activity furthermore leads to increased plasmin activation and a reduced activation of the contact system on the surface of S. pyogenes. All these effects can be reversed when ldh is reintroduced into the mutant via a plasmid. The results demonstrate a previously unappreciated role for LDH in modulation of SpeB maturation.

The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes.

Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced. Accordingly, transcript and proteome analyses revealed decreased expression of several Mga-activated genes. Therefore, and because the sRNA was shown to interact with the 5' UTR of the mga transcript in a gel-shift assay, we designated it MarS for m ga-activating regulatory sRNA. Down-regulation of important virulence factors, including the antiphagocytic M-protein, led to increased susceptibility of the deletion strain to phagocytosis and reduced adherence to human keratinocytes. In a mouse infection model, the marS deletion mutant showed reduced dissemination to the liver, kidney, and spleen. Additionally, deletion of marS led to increased tolerance towards oxidative stress. Our in vitro and in vivo results indicate a modulating effect of MarS on virulence gene expression and on the pathogenic potential of GAS.

EJMS protocol: systematic studies on TiO2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometry-based phosphopeptide stability estimations?

There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.

CIN85 interacting proteins in B cells-specific role for SHIP-1.

The Cbl-interacting 85-kDa protein (CIN85) plays an important role as a negative regulator of signaling pathways induced by receptor tyrosine kinases. By assembling multiprotein complexes this versatile adaptor enhances receptor tyrosine kinase-activated clathrin-mediated endocytosis and reduces phosphatidylinositol-3-kinase-induced phosphatidylinositol-3,4,5-trisphosphate production. Here we report the expression of CIN85 in primary splenic B lymphocytes and the B-lymphoma cell lines WEHI 231 and Ba/F3. Cross-linking of the B cell antigen receptor resulted in an increased association of CIN85 with the ubiquitin ligase Cbl. Through a systematic pull-down proteomics approach we identified 51 proteins that interact with CIN85 in B cells, including proteins not shown previously to be CIN85-associated. Among these proteins, the SH2-containing inositol phosphatase 1 (SHIP-1) co-precipitated with both the full-length CIN85 and each of its three SH3 domains. We also showed that this association is constitutive and depends on a region of 79 amino acids near the carboxyl terminus of SHIP-1, a region rich in potential SH3 domain binding sites. Because SHIP-1 is a major negative regulator of the phosphatidylinositol-3-kinase pathway in lymphocytes, we hypothesize that the interaction between SHIP-1 and CIN85 might synergistically facilitate the down-regulation of phosphatidylinositol-3,4,5-trisphosphate levels.

The cyanobacterial hepatotoxin microcystin binds to proteins and increases the fitness of microcystis under oxidative stress conditions.

Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.

An autoinhibitory domain confers redox regulation to maize glycerate kinase.

Glycerate 3-kinase (GLYK) is the terminal enzyme of the photorespiratory cycle in plants and many cyanobacteria. For several C(4) plants, notably grasses of the NADP-malic enzyme (ME) subtype, redox regulation of GLYK has been reported, but the responsible molecular mechanism is not known. We have analyzed the enzyme from the NADP-ME C(4) plant maize (Zea mays) and found that maize GLYK, in contrast to the enzyme from C(3) plants and a dicotyledonous NADP-ME C(4) plant, harbors a short carboxy-terminal extension. In its oxidized (night) form, a disulfide bridge is formed between the two cysteine residues present in this extra domain, and GLYK activity becomes inhibited. Cleavage of this bond by thioredoxin f produces the fully active thiol form, releasing autoinhibition. Fusion of the maize GLYK redox-regulatory domain to GLYK from C(3) plants confers redox regulation to these otherwise unregulated enzymes. It appears that redox regulation of GLYK could be an exclusive feature of monocotyledonous C(4) plants of the NADP-ME type, in which linear electron transport occurs only in the mesophyll chloroplasts. Hence, we suggest that GLYK, in addition to its function in photorespiration, provides glycerate 3-phosphate for the accelerated production of triose phosphate and its export from the mesophyll. This could facilitate the activation of redox-regulated Calvin cycle enzymes and the buildup of Calvin cycle intermediates in the bundle sheath of these particular C(4) plants during the dark/light transition.

MS characterization of qualitative protein polymorphisms in the spinal cords of inbred mouse strains.

The spinal cord proteomes of two inbred mouse strains with different susceptibility to experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, were investigated by 2-DE and MALDI-MS. A proteome map comprising 304 different protein species was established. Using 2-D fluorescence difference gel electrophoresis, a comparison of the mouse strains revealed 26 qualitatively polymorphic proteins with altered electrophoretic mobility. MS analyses and DNA sequencing were applied to characterize their structural differences and 14 single amino acid substitutions were identified. Moreover, analysis of selectively enriched phosphopeptides from the neurofilament heavy polypeptide of both mouse strains revealed a high degree of diversity in the phosphorylated C-terminal domains of this protein. The described approach is capable to structurally characterize qualitative protein polymorphisms, whereas their functional significance remains to be elucidated. For some proteins formerly associated with experimental autoimmune encephalomyelitis and/or multiple sclerosis structural polymorphisms are described here, which may be subjected to further investigations. In addition, this work should be of general interest for proteomic analysis of inbred strains, because it shows potentials and constraints in the use of 2-DE analysis and MALDI-MS to detect and characterize structural protein polymorphisms.

Erythropoietin receptor-mediated Egr-1 activation: structural requirements and functional implications.

The transcription factor Egr-1 is encoded by an immediate early response gene and has been shown to be a key regulator in the induction of apoptosis, mitogenesis and differentiation. It is rapidly induced by different stimuli including the glycoprotein hormone erythropoietin. In this report, we analyse the role of different erythropoietin receptor substructures for the activation of Egr-1 and the functional consequences of Egr-1 overexpression in the erythroleukemic cell line ELM-I-1. The investigation of receptor variants revealed that the activity of JAK2 and the phosphorylation of receptor tyrosine residues are essential preconditions for the ability to target Egr-1. Furthermore, we observed a close correlation of the abilities of receptors to activate the Ras-MAPK pathway and Egr-1. Using mass spectrometry we identified the Ras-GTPase-activating protein-SH3-domain-binding protein 1 (G3BP-1), a component of the Ras network of proteins, as an Egr-1 interacting protein in EPO stimulated ELM-I-1 cells. The overexpression of Egr-1 in these cells resulted in an enhanced rate of spontaneous erythroid differentiation.

Properties of recombinant glycine decarboxylase P- and H-protein subunits from the cyanobacterium Synechocystis sp. strain PCC 6803.

The multi-enzyme complex glycine decarboxylase is important for one-carbon metabolism, essential for the photorespiratory glycolate cycle of plants, and comprises four different polypeptides, P-, H-, T-, and L-protein. We report on the production and properties of recombinant P-protein from the cyanobacterium Synechocystis and also describe features of recombinant H-protein from the same organism. The P-protein shows enzymatic activity with lipoylated H-protein and very low activity with H-apoprotein or lipoate as artificial cofactors. Its affinity towards glycine is unaffected by the presence and nature of the methyleneamine acceptor molecule. The cyanobacterial H-protein apparently forms stable dimers.

2-DE profiling of GDNF overexpression-related proteome changes in differentiating ST14A rat progenitor cells.

Targeted differentiation of neural progenitor cells (NPCs) is a challenge for treatment of neurodegenerative diseases by cell replacement therapy and cell signalling manipulation. Here, we applied a proteome profiling approach to the rat striatal progenitor model cell line ST14A in order to elucidate cellular differentiation processes. Native cells and cells transfected with the glial cell line-derived neurotrophic factor (GDNF) gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. 2-DE combined with MALDI-MS was used to create a reference 2-DE-map of 652 spots of which 164 were identified and assigned to 155 unique proteins. For identification of protein expression changes during cell differentiation, spot patterns of triplicate gels were matched to the 2-DE-map. Besides proteins that display expression changes in native cells, we also noted 43 protein-spots that were differentially regulated by GDNF overexpression in more than four time points of the experiment. The expression patterns of putative differentiation markers such as annexin 5 (ANXA5), glucosidase II beta subunit (GLU2B), phosphatidylethanolamine-binding protein 1 (PEBP1), myosin regulatory light chain 2-A (MLRA), NASCENT polypeptide-associated complex alpha (NACA), elongation factor 2 (EF2), peroxiredoxin-1 (PRDX1) and proliferating cell nuclear antigen (PCNA) were verified by Western blotting. The results reflect the large rearrangements of the proteome during the differentiation process of NPCs and their strong modification by neurotrophic factors like GDNF.

Proteomic analysis of the E2F1 response in p53-negative cancer cells: new aspects in the regulation of cell survival and death.

E2F1 is an essential transcription factor that regulates cell-cycle progression and apoptosis. Overexpression of E2F1 sensitizes neoplastic cells to apoptosis and leads to tumor growth suppression, making it an interesting target for anticancer therapy. Use of E2F1 as a therapeutic, however, requires a detailed knowledge of the mechanisms by which it controls cellular proliferation and apoptosis, and of other potential E2F1 activities. In this study, a differential proteome analysis was performed to identify proteins associated with E2F1 activity in inducible p53-deficient Saos-2ERE2F1 osteosarcoma cells. 2-DE revealed a distinct protein profile at 32 h after E2F1 activation. Thirty-three proteins were reproducibly identified as either up-regulated or down-regulated. Proteins were identified by MALDI-MS. They included hitherto unknown E2F1 target proteins of cytoskeletal origin, chaperones, enzymes, proteasomal proteins, and several heterogeneous nuclear ribonucleoproteins, suggesting its role in the ER-stress response, protein degradation, and modulation of pre-mRNA splicing. Protein analysis-derived results were verified by Western blot using representative protein candidates. Thirteen identified proteins were the products of genes known to be cancer related. Thus, proteome analysis provides new information about the complexity of E2F1 activities in human cancer cells that may be considered when using E2F1 as a drug.

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM).

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.

Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences.

A proteomic approach has been used to establish a proteome map and differentiate between the protein composition of tonsils from patients with chronic tonsillitis (CT) and that of tonsils with hyperplasia (HPL). Two-dimensional gel analysis was performed with material from four patients with HPL and five patients with CT. An average of approximately 600 spots were detected in each gel. A total of 127 different proteins were identified in 158 spots analyzed by mass spectrometry. Our study revealed disease-associated differences between protein abundance for two protein spots, an HSP27 isoform and UMP-CMP kinase. Both protein spots were more abundant in the CT group. HSP27 ELISA was performed for 32 patients, 12 belonging to the HPL group and 20 to the CT group. ELISA could not be used to differentiate HSP27 isoforms nor to distinguish CT from HPL. HSP27 was found to migrate to two further protein spots in the 2D gels. The differently expressed HSP27 isoform migrated as the most acidic of all the HSP27 isoforms detected, indicating the highest degree of phosphorylation. The sum of all three HSP27 abundances in the gels from the CT group was not different from that of the HPL group, consistent with the ELISA results. Our results suggest that phosphorylation differences caused the observed migration differences of HSP27. Together with the UMP-CMP kinase abundance differences, we conclude that kinase and/or phosphatase activity are different in CT and HPL.

Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis.

Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels.

Matrix-assisted laser desorption/ionization- quadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes.

Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.