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.

Digestive Physiology of Octopus maya and O. mimus: Temporality of Digestion and Assimilation Processes.

Digestive physiology is one of the bottlenecks of octopus aquaculture. Although, there are successful experimentally formulated feeds, knowledge of the digestive physiology of cephalopods is fragmented, and focused mainly on Octopus vulgaris. Considering that the digestive physiology could vary in tropical and sub-tropical species through temperature modulations of the digestive dynamics and nutritional requirements of different organisms, the present review was focused on the digestive physiology timing of Octopus maya and Octopus mimus, two promising aquaculture species living in tropical (22-30°C) and sub-tropical (15-24°C) ecosystems, respectively. We provide a detailed description of how soluble and complex nutrients are digested, absorbed, and assimilated in these species, describing the digestive process and providing insight into how the environment can modulate the digestion and final use of nutrients for these and presumably other octopus species. To date, research on these octopus species has demonstrated that soluble protein and other nutrients flow through the digestive tract to the digestive gland in a similar manner in both species. However, differences in the use of nutrients were noted: in O. mimus, lipids were mobilized faster than protein, while in O. maya, the inverse process was observed, suggesting that lipid mobilization in species that live in relatively colder environments occurs differently to those in tropical ecosystems. Those differences are related to the particular adaptations of animals to their habitat, and indicate that this knowledge is important when formulating feed for octopus species.

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.

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.