Targeting TNFα in severe psoriasis-mass spectrometry reveals a time-dependent specific inhibition of Factor H in responding patients.

Drugs targeting TNFα (eg, Etanercept®) provide effective control of severe psoriasis. In absence of validated biological parameters of inflammation in psoriasis most decisions on therapeutics have relied mostly on clinical criteria, namely the "Psoriasis Area and Severity Index" (PASI). The purpose of this study was to assess by mass spectrometry alterations in concentrations of serum proteins that specifically correlated with effectiveness of Etanercept treatment. This prospective study enrolled 10 patients suffering from moderate to severe psoriasis (PASI score > 10 and < 17) and treated with Etanercept over a period of 24 weeks; 10 healthy, age-matched volunteers provided controls. Serum proteins sensitive to Etanercept treatment were identified using SELDI-TOF (surface-enhanced laser desorption and ionization - time of flight) coupled to nano LC-ESI/MS (nano liquid chromatography-electrospray ionization/tandem mass spectrometry) technologies. For comparisons between groups of individuals p-values (considered significant when < 0.01) were estimated with non-parametric tests, namely Mann-Whitney (for unpaired data) and Wilcoxon signed-rank (for paired data). In responding patients it could be shown using SELDI-TOF spectrometry that two proteins (134 kDa and 4.3 kDa) return to control levels by 24 weeks of treatment. Using nano LC-ESI/MS the 134 kDa species was identified as complement Factor H. These observations deserve further analyses utilizing larger cohorts of patients. Determination of Factor H levels may become a complementary tool to follow remission or predict the onset of relapse in the follow-up of patients under treatment with Etanercept.

A meta-analysis to evaluate the cellular processes regulated by the interactome of endogenous and over-expressed estrogen receptor alpha.

The nature of the proteins complexes that regulate ERα subcellular localization and activity is still an open question in breast cancer biology. Identification of such complexes will help understand development of endocrine resistance in ER+ breast cancer. Mass spectrometry (MS) has allowed comprehensive analysis of the ERα interactome. We have compared six published works analyzing the ERα interactome of MCF-7 and HeLa cells in order to identify a shared or different pathway-related fingerprint. Overall, 806 ERα interacting proteins were identified. The cellular processes were differentially represented according to the ERα purification methodology, indicating that the methodologies used are complementary. While in MCF-7 cells, the interactome of endogenous and over-expressed ERα essentially represents the same biological processes and cellular components, the proteins identified were not over-lapping; thus, suggesting that the biological response may differ as the regulatory/participating proteins in these complexes are different. Interestingly, biological processes uniquely associated to ERα over-expressed in HeLa cell line included L-serine biosynthetic process, cellular amino acid biosynthetic process and cell redox homeostasis. In summary, all the approaches analyzed in this meta-analysis are valid and complementary; in particular, for those cases where the processes occur at low frequency with normal ERα levels, and can be identified when the receptor is over-expressed. However special effort should be put into validating these findings in cells expressing physiological ERα levels.

An integrated perspective and functional impact of the mitochondrial acetylome.

Growing evidence suggests that a range of reversible protein post-translational modifications such as acetylation regulates mitochondria signalling, impacting cellular homeostasis. However, the extent of this type of regulation in the control of mitochondria functionality is just beginning to be discovered, aided by the availability of high-resolution mass spectrometers and bioinformatic tools. Data mining from literature on protein acetylation profiling focused on mitochondria isolated from tissues retrieved more than 1395 distinct proteins, corresponding to more than 4858 acetylation sites. ClueGo analysis of identified proteins highlighted oxidative phosphorylation, tricarboxylic acid cycle, fatty acid oxidation and amino acid metabolism as the biological processes more prone to regulation through acetylation. This review also examines the physiological relevance of protein acetylation on the molecular pathways harbored in mitochondria under distinct pathophysiological conditions as caloric restriction and alcohol-induced liver damage. This integrative perspective will certainly help to envisage future studies targeting the regulation of mitochondrial functionality.

One decade of salivary proteomics: current approaches and outstanding challenges.

Efforts have been made in the last decade towards the complete characterization of saliva proteome using gel-based and gel-free approaches. The combination of these strategies resulted in the increment of the dynamic range of saliva proteome, which yield in the identification of more than 3,000 different protein species. Comparative protein profiling using isotope labeling and label free approaches has been used for the identification of novel biomarkers for oral and related diseases. Although progresses have been made in saliva proteome characterization, the comparative profiling in different pathophysiological conditions is still at the beginning if compared to other bodily fluids. The potential biomarkers identified so far lack specificity once common differentially expressed proteins were detected in the saliva of patients with distinct diseases. In addition, recent research works focused on saliva peptidome profiling already allowed a better understanding of peptides' physiological role in oral cavity. This review provides an overview of the major achievements in saliva proteomics giving emphasis to methodological concerns related with saliva collection, treatment and analysis, as well as the main advantages and pitfalls underlying salivary proteomic strategies and potential clinical outcomes.

Identification of salivary proteins at oil-water interfaces stabilized by lysozyme and beta-lactoglobulin.

In this research, we investigated the interaction occurring between oil-in-water emulsion droplets, stabilized by different emulsifiers, i.e. lysozyme and beta-lactoglobulin (beta-lg), and salivary proteins (SPs) with a molecular mass (M(r)) above about 10kDa. Different techniques, i.e. infrared spectroscopy, Western blotting, PAS staining and SDS-PAGE coupled to MS, were employed for this purpose. This study demonstrated the interaction between several salivary proteins and the emulsifiers at the oil-water interfaces. In particular, results show that the high M(r) mucin MUC5B was strongly bound to lysozyme stabilized emulsions, whereas beta-lg stabilized emulsions associated with MUC7 and, moderately, with MUC5B. Furthermore, we observed that salivary proteins in the range M(r) 10-100kDa associated differently with emulsion droplets. A large majority of SPs was found to interact with lysozyme stabilized emulsion droplets whilst in case of beta-lg stabilized emulsions, the SPs distribute more evenly between the fraction associated and non-associated with the droplets. A clear example is alpha-amylase (M(r) approximately 55kDa) which predominantly associates with lysozyme stabilized emulsion droplets, but not with beta-lg emulsion droplets. To conclude, our findings indicate that adsorption/association of salivary protein components onto the emulsion droplets is related to the type of emulsifying proteins at the oil-water interfaces and it is probably driven by the overall net charge at the droplet's oil-water interfaces, i.e. positive for lysozyme stabilized emulsions and negative for beta-lactoglobulin stabilized emulsion at neutral pH.

Detection and characterization of cyclic hydroxylamine adducts by mass spectrometry.

Two cyclic hydroxylamines (cHA) bearing pyrrolidine (CPH) and piperidine moieties (TMTH) were evaluated to trap hydroxyl, peptide and phospholipid free radicals using mass spectrometry for their detection. The cHA ionized as [M+H](+) ions, showing higher relative abundances when compared to the DMPO, probably due to higher ionization efficiency. In the presence of hydroxyl radicals, both cHA generated new ions that could be attributed to loss of (*)H and (*)CH(3), most likely deriving from decomposition reactions of the nitroxide spin adduct. Addition of cHA to Leucine-enkephalin and palmitoyl-lineloyl-glycerophosphatidylcholine free radicals promoted the formation of cHA biomolecule adducts, which were confirmed by MS/MS data. Results suggest that the cHA are not suitable for hydroxyl radical trapping but can be used for trapping biomolecule radicals, having the advantage, over the most used cyclic nitrones, of being water soluble. The biomolecule adducts identified by MS are ESR silent, evidencing the importance of MS detection.

Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis.

We studied 82 Portuguese individuals, 57 with hereditary spherocytosis (HS) and 25 unaffected controls. We performed standardized diagnosis tests, including electrophoretic membrane protein analysis to identify and quantify protein deficiencies underlying HS. Membrane bound hemoglobin (MBH) and band 3 profiles were determined as oxidative stress and aging markers. A protein of about 22 kDa, present in 21 of 57 HS patients, but not in controls, was identified as peroxiredoxin 2 (Prx2), by mass-spectroscopy and by immunoblotting. Human erythrocyte Prx2 is a peroxiredoxin with thiol-specific antioxidant activity. The presence of Prx2 in erythrocyte membranes was linked to higher levels of oxidative stress, as reflected by significantly increased MBH in those HS patients. No relation with HS clinical severity was observed and Prx2 was detected in all types of membrane protein abnormalities. Prx2 membrane linkage is associated with a higher oxidative stress susceptibility of HS erythrocytes.

Peptide profile of human acquired enamel pellicle using MALDI tandem MS.

The present study proposes a strategy for human in vivo acquired enamel pellicle (AEP) peptidome characterisation based on sequential extraction with guanidine and TFA followed by MALDI-TOF/TOF identification. Three different nanoscale analytical approaches were used: samples were subjected to tryptic digestion followed by nano-HPLC and mass spectrometry (MS and MS/MS) analysis. Undigested samples were analysed by LC-MS (both linear and reflector modes) and LC-MS/MS analysis, and samples were subjected to nano-HPLC followed by on-plate digestion and mass spectrometry (MS and MS/MS) analysis. The majority of the identifications corresponded to peptide/protein fragments of salivary protein, belonging to the classes: acidic PRPs, basic PRPs, statherin, cystatins S and SN and histatin 1 (all also identified in intact form). Overall, more than 90 peptides/proteins were identified. Results clearly show that peptides with acidic groups are enriched in the TFA fraction while peptides with no acidic or phosphate groups are prevalent on the guanidine extract. Also, phosphorylated peptides were observed mainly on the TFA fraction. Fragments present in the AEP show a predominance of cleavage points located at Arg, Tyr and Lys residues. Obtained data suggest that proteolytic activity could influence AEP formation and composition.

Subcellular proteomics of mice gastrocnemius and soleus muscles.

A proteomics characterization of mice soleus and gastrocnemius white portion skeletal muscles was performed using nuclear, mitochondrial/membrane, and cytosolic subcellular fractions. The proposed methodology allowed the elimination of the cytoskeleton proteins from the cytosolic fraction and of basic proteins from the nuclear fraction. The subsequent protein separation by two-dimensional gel electrophoresis prior to mass spectrometry analysis allowed the detection of more than 600 spots in each muscle. In the gastrocnemius muscle fractions, it was possible to identify 178 protein spots corresponding to 108 different proteins. In the soleus muscle fractions, 103 different proteins were identified from 253 positive spot identifications. A bulk of cytoskeleton proteins such as actin, myosin light chains, and troponin were identified in the nuclear fraction, whereas mainly metabolic enzymes were detected in the cytosolic fraction. Transcription factors and proteins associated with protein biosynthesis were identified in skeletal muscles for the first time by proteomics. In addition, proteins involved in the mitochondrial redox system, as well as stress proteins, were identified. Results confirm the potential of this methodology to study the differential expressions of contractile proteins and metabolic enzymes, essential for generating functional diversity of muscles and muscle fiber types.

Peptidomic analysis of human acquired enamel pellicle.

Human acquired enamel pellicle is the result of a selective interaction of salivary proteins and peptides with the tooth surface. In the present work, the characterization of the peptides as well as the type of interactions established with the enamel surface was performed. Peptides from in vivo bovine enamel implants in the human oral cavity were sequentially extracted using guanidine and trifluoroacetic acid solutions and the fractions obtained were analysed by LC-MS and LC-MS/MS. Based on the LC-MS data, six phosphorylated peptides were identified in an intact form, strongly adsorbed to the enamel surface. Data from the LC-MS/MS analyses allowed us to identified 30 fragment peptides non-covalently bonded to enamel [basic proline-rich proteins, histatins (1 and 3) and acidic proline-rich protein classes]. The tandem mass spectrometry experiments showed the existence of a pattern of amide bond cleavage for the different identified peptide classes suggesting a selective proteolytic activity. For histatins, a predominance of cleavage at Arg, Lys and His residues was observed, while for basic proline-rich proteins, cleavage at Arg and Pro residues prevailed. In the case of acidic proline-rich proteins, a clearly predominance of cleavage of the Gln-Gly amide bond was evident.

Two-dimensional electrophoresis study of in vitro pellicle formation and dental caries susceptibility.

In the present study, a proteomic approach was applied to evaluate the influence of salivary protein composition on in vitro dental pellicle formation and its possible correlation with dental caries. Whole saliva, collected from caries-free and caries-susceptible subjects, was analyzed by two-dimensional electrophoresis, and protein spots were identified by mass spectrometry. Data analysis of salivary protein composition showed a statistically significant correlation between the quantity of acidic proline-rich proteins (PRPs), lipocalin, cystatin SN and cystatin S, and samples from the caries-free group of subjects [decayed, missing or filled teeth (DMFT) = 0]. Samples from subjects with a high DMFT index appear to be correlated with high levels of amylase, immunoglobulin A, and lactoferrin. In vitro pellicle-composition experiments showed the same correlations found for whole saliva. As cystatins are known physiological inhibitors of cathepsins, the higher quantities of lipocalin, and cystatins S and SN found in the samples from the caries-free subjects suggest that inhibition of proteolytic events on other salivary proteins may indirectly provide tooth protection. The correlation between higher levels of the phosphorylated acidic PRPs 1/2 with samples from the caries-free group also suggests a protective role for these proteins.

The role of salivary peptides in dental caries.

Dental caries is a complex disease, characterized by demineralization of tooth structure. With a protective role, several salivary phosphopeptides appear to be involved in remineralization processes, delaying the loss of tooth structure. In this work we have correlated peptide saliva composition with dental caries susceptibility through the analysis of saliva and hydroxyapatite-adsorbed salivary peptides samples. Saliva samples were obtained from two groups, a caries-free and a cariessusceptible group, and were analysed using HPLC-MS and a sequential extraction with 6 m of guanidine followed by tri fluoroacetate. Data analysis has allowed us to verify a strong correlation between large amounts phosphopeptides (PRP1/3, histatin 1 and statherin), and the absence of dental caries, which reinforces the importance of these peptides in the maintenance of tooth integrity. In addition, in the caries-susceptible group a high number of peptide fragments was observed, suggesting a high proteolytic activity.

Separation of peroxidation products of diacyl-phosphatidylcholines by reversed-phase liquid chromatography-mass spectrometry.

Lipid peroxidation process has attracted much attention due to the growing evidence of its involvement in the pathogenesis of age-related diseases. The monitoring of the lipid peroxidation products in phospholipids, formed under oxidative stress conditions, may provide new markers for oxidative stress signaling and for disease states, giving new insights in the pathogenesis process. Reversed-phase liquid chromatographic method coupled to mass spectrometry was developed for the separation of oxidized glycero-phosphatidylcholine (GPC) peroxidation products formed by the Fenton reaction that mimic in vivo oxidative stress conditions. The LC-MS conditions were applied for the separation of peroxidation products of oleoyl- (POPC), lineloyl- (PLPC) and arachidonoyl-palmitoyl phosphatidylcholine (PAPC). The peroxidation products separated included products resulting from the insertion of oxygen atoms in the sn-2 chain (long-chain), and products with the sn-2 chain shortened resulting from cleavage of oxygen-centered radicals (short-chain). Among long-chain products were the keto, hydroxy, hydroperoxide and poly-hydroxy derivatives, while short-chain products included dicarboxylic acids, aldehydes and hydroxy-aldehydes. Separation of long-chain products formed in each phosphatidylcholine was observed, and the reconstructed ion chromatogram of each ion showed an increase in the number of peaks with the increase in the number of oxygen atoms inserted into the phospholipid. Separation of short-chain products took place according to the functional group present at the sn-2 moiety that allowed the elution of dicarboxylic acids distinct from aldehydes. Separation between isomeric structures that were present in short- and long-chain products was also achieved.

Analysis of salivary peptides using HPLC-electrospray mass spectrometry.

Salivary peptides are involved in a wide range of functions constituting the first line of defence of oral cavity and precursors of dental pellicle formation. The presence of mucins in saliva makes difficult the analysis of the proteic content. This is due mainly to aggregation phenomenon between mucins and other high molecular weight glycoproteins and salivary proteins. Considering the importance of salivary peptides in biological functions, we have evaluated the influence of four different extraction methodologies on the separation and identification of these proteins by HPLC-MS. Based on their molecular weight, we identified a total of 22 peptides when extraction was performed using a solution of guanidine (6 m), compared with 14 peptides identified when saliva is acidified with TFA, which is an often used procedure. Our results also show the presence of mucin bind peptides, which include statherin, PRP1, PRP3, Histatin 1 and Histatin 5.

In vitro hydroxyapatite adsorbed salivary proteins.

In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified.

Identification of human whole saliva protein components using proteomics.

The determination of salivary biomarkers as a means of monitoring general health and for the early diagnosis of disease is of increasing interest in clinical research. Based on the linkage between salivary proteins and systemic diseases, the aim of this work was the identification of saliva proteins using proteomics. Salivary proteins were separated using two-dimensional (2-D) gel electrophoresis over a pH range between 3-10, digested, and then analyzed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Proteins were identified using automated MS and MS/MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. Ninety spots give identifications with high statistical reliability. Of the identified proteins, 11 were separated and identified in saliva for the first time using proteomics tools. Moreover, three proteins that have not been previously identified in saliva, PLUNC, cystatin A, and cystatin B were identified.

Detection and characterization by mass spectrometry of radical adducts produced by linoleic acid oxidation.

The formation of linoleic acid radical species under the oxidative conditions of the Fenton reaction (using hydrogen peroxide and Fe (II)) was monitored by FAB-MS and ES-MS using the spin trap 5,5-dimethyl-1-pyrrolidine-N-oxide, DMPO. Both the FAB and ES mass spectra were very similar and showed the presence of ions corresponding to carbon- and oxygen centered spin adducts (DMPO/L*, DMPO/LO*, and DMPO/LOO*). Cyclic structures, formed between the DMPO oxygen and the neighboring carbon of the fatty acid, were also observed. Electrospray tandem mass spectrometry of these ions was performed to confirm the proposed structure of these adducts. All MS/MS spectra showed an ion at m/z 114, correspondent to the [DMPO + H]+, and a fragment ion due to loss of DMPO (loss of 113 Da), confirming that they are DMPO adducts. ES-MS/MS spectra of alkoxyl radical adducts (DMPO/LO*) showed an additional ion at m/z 130 [DMPO - O + H]+, while ES MS/MS of peroxyl radical adducts (DMPO/LOO*) showed a fragment ion at m/z 146 [DMPO - OO + H]+, confirming both structures. Other fragment ions were observed, such as alkyl acylium radical ions, formed by cleavage of the alkyl chain after loss of water and the DMPO molecule. The identification of fragment ions observed in the MS/MS spectra of the different DMPO adducts suggests the occurrence of structural isomers containing the DMPO moiety both at C9 and C13. The use of ES tandem mass spectrometry, associated with spin trapping experiments, has been shown to be a valuable tool for the structural characterization of carbon and oxygen-centered spin adducts of lipid radicals.

Identification of oxidation products and free radicals of tryptophan by mass spectrometry.

New oxidation products and free radicals derived from tryptophan (Trp) oxidation under Fenton reaction conditions were identified using mass spectrometry. After the oxidation of tryptophan using hydrogen peroxide and iron (II) system (Fenton reaction), mono- and dihydoxy tryptophans and N-formylkynurenine were identified using electrospray mass spectrometry (ES-MS) and ES-MS/MS. Besides these products, new products resulting from the reaction of tryptophan and oxidized tryptophan and 3-methyl indole derivatives were also identified. The 3-methyl indole derivatives resulted, most probably, from the oxidation process and not from in-source processes. A dimer formed by cross-linking between two Trp radicals (Trp-Trp), similar to the previously described tyrosine dimer was observed, as well as the corresponding monohydroxy-dimer (Trp-Trp-OH). Tandem mass spectrometry was used to identify the structures of these new oxidation products. Free radicals derived from tryptophan oxidation under Fenton reaction were detected using as spin trap the DMPO. The free radical species originated during the oxidation reaction formed stable adducts with the spin trap, and these adducts were identified by ES-MS. New adducts of oxidized tryptophan radicals, namely monohydroxy-tryptophan and dihydroxy-Trp dimer radicals, with one and two DMPO spin trap molecules where identified. Tandem mass spectrometry was used to confirm the proposed structure of the observed adducts.