Personalized rheumatic medicine through dose reduction reduces the cost of biological treatment - a retrospective intervention analysis.

Objective: The effects of a dose-reduction intervention of biological disease-modifying anti-rheumatic drugs (bDMARDs) in patients in remission were analysed with epidemiology and health economics strategies. The aims were to analyse changes in bDMARD dosage, evaluate potential disease worsening, and estimate cost reduction. Method: This uncontrolled single-centre observational study analysed bDMARD-treated patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and spondyloarthritis (SpA). bDMARD expenditure constituted a proxy for bDMARD doses, which enabled group-level analysis. Interrupted time-series regression was used to analyse changes in treatment cost due to the dose reduction. Disease activity and treatment durations were monitored to investigate disease worsening. Results: In total, 997 biological treatment cases were analysed. This involved 527 bDMARD patients, where an unknown fraction of patients was given reduced doses. Disease activity of RA and PsA patients decreased from 2001 to 2009 and remained stable after that, while disease activity for SpA patients was unchanged, indicating no disease worsening from the intervention. The dose tapering resulted in decreased bDMARD expenditure, indicating a decrease in bDMARD consumption, which led to an accumulated cost reduction of 4 178 000 EUR. Conclusions: The results suggest that dose reduction can be safely performed in patients in treatment remission on a group level without compromising treatment efficacy. Subcutaneous bDMARDs, including abatacept, adalimumab, and etanercept, were observed to be well suited to customizing dosage. This study highlights the potential for individualized and personalized rheumatic medicine by providing dose reduction to individual patients, while monitoring disease activity.

Presence of HLA-DR Molecules and HLA-DRB1 mRNA in Circulating CD4(+) T Cells.

The human major histocompatibility complex class II isotype HLA-DR is currently used as an activation marker for T cells. However, whether an endogenous protein expression or a molecular acquisition accounts for the presence of HLA-DR on T cells remains undetermined and still controversial. To further characterize this phenomenon, we compared several aspects of the presence of the HLA-DR protein to the presence of associated mRNA (HLA-DRB1), focusing on human T cells from peripheral blood of healthy individuals. Using a flow cytometric approach, we determined that the HLA-DR observed on CD4(+) T cells was almost exclusively cell surface-associated, while for autologous CD19(+) B cells, the protein could be located in the plasma membrane as well as in the cytoplasm. Moreover, negligible expression levels of HLA-DRB1 were found in CD4(+) T cells, using an HLA-DRB1 allele-specific qPCR assay. Finally, the presence of HLA-DR was not confined to activated CD4(+) and CD8(+) T cells, as evaluated by the co-expression of CD25. The functional role of the HLA-DR molecule on T cells remains enigmatic; however, this study presents evidence of fundamental differences for the presence of HLA-DR on T cells from HLA-DR in the context of antigen-presenting cells, which is a well-known phenomenon. Although an inducible endogenous protein expression cannot be excluded for the T cells, our findings suggest that a re-evaluation of the HLA-DR as a T cells activation marker is warranted.

Digging into the extracellular matrix of a complex microbial community using a combined metagenomic and metaproteomic approach.

Knowledge about identity and function of extracellular polymeric substances (EPS) in complex microbial communities is sparse, although these components have a large influence on the function of the microbial communities. We investigated the presence of selected genes potentially involved in EPS production in a 145 Mbp metagenome prepared by Illumina sequencing from a full-scale wastewater treatment plant carrying out enhanced biological phosphorus removal (EBPR). A range of genes involved in alginate production was identified and assigned mainly to bacteria from the phylum Bacteroidetes. Furthermore, several proteins in the EPS matrix were extracted, purified and identified by mass spectrometry. By using the metagenome as a reference for the metaproteomic analysis, more proteins were identified compared to using only publicly available databases. This illustrates the low degree of similarity between the bacteria in the EBPR community and the sequenced bacteria in the public databases. Hence, the combination of metagenomics and metaproteomics presented here is needed to investigate the identity of the proteins in the EPS matrix.

Quantitative proteomic analysis of ibuprofen-degrading Patulibacter sp. strain I11.

Ibuprofen is the third most consumed pharmaceutical drug in the world. Several isolates have been shown to degrade ibuprofen, but very little is known about the biochemistry of this process. This study investigates the degradation of ibuprofen by Patulibacter sp. strain I11 by quantitative proteomics using a metabolic labelling strategy. The whole-genome of Patulibacter sp. strain I11 was sequenced to provide a species-specific protein platform for optimal protein identification. The bacterial proteomes of actively ibuprofen-degrading cells and cells grown in the absence of ibuprofen was identified and quantified by gel based shotgun-proteomics. In total 251 unique proteins were quantitated using this approach. Biological process and pathway analysis indicated a number of proteins that were up-regulated in response to active degradation of ibuprofen, some of them are known to be involved in the degradation of aromatic compounds. Data analysis revealed that several of these proteins are likely involved in ibuprofen degradation by Patulibacter sp. strain I11.

Effect of minor milk proteins in chymosin separated whey and casein fractions on cheese yield as determined by proteomics and multivariate data analysis.

The objective of this work was to find regressions between minor milk proteins or protein fragments in the casein or sweet whey fraction and cheese yield because the effect of major milk proteins was evaluated in a previous study. Proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry in combination with multivariate data analysis were used to study the effect of variations in milk protein composition in chymosin separated whey and casein fractions on cheese yield. By mass spectrometry, a range of proteins significant for the cheese yield was identified. Among others, a C-terminal fragment of beta-casein had a positive effect on the cheese yield expressed as grams of cheese per 100 g of milk, whereas several other minor fragments of beta-, alpha(s1)-, and alpha(s2)-casein had positive effects on the transfer of protein from milk to cheese. However, the individual effect of each identified protein was relatively low. Therefore, further studies of the relations between different proteins/peptides in the rennet casein or sweet whey fractions and cheese yield are needed for advanced understanding and prediction of cheese yield.

Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface.

Identification and detailed characterization of complex mixtures of proteins separated by polyacrylamide gel electrophoresis (PAGE) require optimized and robust methods for interfacing electrophoretic techniques to mass spectrometry. Peptide mapping by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used as the first protein screening method in many laboratories because of its inherent simplicity, mass accuracy, sensitivity and relatively high sample throughput. We present a simplified sample preparation method for MALDI-MS that enables in-gel digestion of protein samples directly on the MALDI-MS metal probe. Removal of detergent and reagents as well as protein reduction and S-alkylation were performed prior to cutting of protein samples from the polyacrylamide gel slab. The general utility of this approach was demonstrated by on-probe digestion and MALDI-MS peptide mapping of femtomole amounts of standard proteins isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A representative set of 47 human proteins obtained from a silver stained two-dimensional electrophoretic gel was analyzed by the new method and resulted in a success rate for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation protocol while being less labour intensive and more cost-effective due to minimal consumption of reagents, enzymes and consumables. Preliminary data obtained on a MALDI quadrupole-TOF tandem mass spectrometer demonstrated the utility of the on-probe digestion protocol for peptide mass mapping and peptide sequencing on this instrument. Automation of the on-probe protein digestion procedure and its combination with automated MALDI tandem mass spectrometry should be advantageous in proteomics research aimed at the systematic identification and analysis of large sets of proteins from electrophoretic gels.

Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis.

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectrospray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nanoelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine beta-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 beta subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis.

Electron capture dissociation of singly and multiply phosphorylated peptides.

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.

Binding of 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947).

14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription. The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H(+)-ATPase. The 14-3-3 binding site in the Arabidopsis plasma membrane H(+)-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr(946)-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947). We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.