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Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microbial species in clinical microbiology laboratories. MALDI-TOF-MS has swiftly become the new gold-standard method owing to its key advantages of simplicity and robustness. However, as with all new methods, adoption of the MALDI-TOF MS approach is still not widespread. Optimal sample preparation has not yet been achieved for several applications, and there are continuing discussions on the need for improved database quality and the inclusion of additional microbial species. New applications such as in the field of antimicrobial susceptibility testing have been proposed but not yet translated to the level of ease and reproducibility that one should expect in routine diagnostic systems. Finally, during routine identification testing, unexpected results are regularly obtained, and the best methods for transmitting these results into clinical care are still evolving. We here discuss the success of MALDI-TOF MS in clinical microbiology and highlight fields of application that are still amenable to improvement.

Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has entered clinical diagnostics and is today a generally accepted and integral part of the workflow for microbial identification. MALDI-TOF MS identification systems received approval from national and international institutions, such as the USA-FDA, and are continuously improved and adopted to other fields like veterinary and industrial microbiology. The question is whether MALDI-TOF MS also has the potential to replace other conventional and molecular techniques operated in routine diagnostic laboratories. Areas covered: We give an overview of new advancements of mass spectral analysis in the context of microbial diagnostics. In particular, the expansion of databases to increase the range of readily identifiable bacteria and fungi, the refined discrimination of species complexes, subspecies, and types, the testing for antibiotic resistance or susceptibility, progress in sample preparation including automation, and applications of other mass spectrometry techniques are discussed. Expert opinion: Although many new approaches of MALDI-TOF MS are still in the stage of proof of principle, it is expectable that MALDI-TOF MS will expand its role in the clinical microbiology laboratory of the future. New databases, instruments and analytical software modules will continue to be developed to further improve diagnostic efficacy.

International audience; Resistance to β-lactams in Acinetobacter baumannii involves various mechanisms. To decipher them, whole genome sequencing (WGS) and real-time quantitative polymerase chain reaction (RT-qPCR) were complemented by mass spectrometry (MS) in selected reaction monitoring mode (SRM) in 39 clinical isolates. The targeted label-free proteomic approach enabled, in one hour and using a single method, the quantitative detection of 16 proteins associated with antibiotic resistance: eight acquired β-lactamases (i.e. GES, NDM-1, OXA-23, OXA-24, OXA-58, PER, TEM-1, and VEB), two resident β-lactamases (i.e. ADC and OXA-51-like) and six components of the two major efflux systems (i.e. AdeABC and AdeIJK). Results were normalized using "bacterial quantotypic peptides," i.e. peptide markers of the bacterial quantity, to obtain precise protein quantitation (on average 8.93% coefficient of variation for three biological replicates). This allowed to correlate the levels of resistance to β-lactam with those of the production of acquired as well as resident β-lactamases or of efflux systems. SRM detected enhanced ADC or OXA-51-like production and absence or increased efflux pump production. Precise protein quantitation was particularly valuable to detect resistance mechanisms mediated by regulated genes or by overexpression of chromosomal genes. Combination of WGS and MS, two orthogonal and complementary techniques, allows thereby interpretation of the resistance phenotypes at the molecular level.

Number of transitions and peptides per protein and the percent of missing and zero values among protein concentration measurements. (DOCX 18Â kb)

The full details for sample preparation and SRM analysis. (DOCX 16Â kb)

Resistance to β-lactams in Acinetobacter baumannii involves various mechanisms. To decipher them, whole genome sequencing (WGS) and real-time quantitative polymerase chain reaction (RT-qPCR) were complemented by mass spectrometry (MS) in selected reaction monitoring mode (SRM) in 39 clinical isolates. The targeted label-free proteomic approach enabled, in one hour and using a single method, the quantitative detection of 16 proteins associated with antibiotic resistance: eight acquired β-lactamases (i.e. GES, NDM-1, OXA-23, OXA-24, OXA-58, PER, TEM-1, and VEB), two resident β-lactamases (i.e. ADC and OXA-51-like) and six components of the two major efflux systems (i.e. AdeABC and AdeIJK). Results were normalized using “bacterial quantotypic peptides,” i.e. peptide markers of the bacterial quantity, to obtain precise protein quantitation (on average 8.93% coefficient of variation for three biological replicates). This allowed to correlate the levels of resistance to β-lactam with those of the production of acquired as well as resident β-lactamases or of efflux systems. SRM detected enhanced ADC or OXA-51-like production and absence or increased efflux pump production. Precise protein quantitation was particularly valuable to detect resistance mechanisms mediated by regulated genes or by overexpression of chromosomal genes. Combination of WGS and MS, two orthogonal and complementary techniques, allows thereby interpretation of the resistance phenotypes at the molecular level.

International audience; The cells in an organism emit different amounts of proteins according to their clinical state (healthy/pathological, for instance). The resulting proteomic profile can be used for early detection, diagnosis, and therapy planning. In this paper, we study the classification of a proteomic sample from the point of view of an inverse problem with a joint Bayesian solution, called inversion-classification. We propose a hierarchical physical forward model and present encouraging results from both simulation and clinical data.

International audience; This paper addresses the question of biomarker discovery in proteomics. Given clinical data regarding a list of proteins for a set of individuals, the tackled problem is to extract a short subset of proteins the concentrations of which are an indicator of the biological status (healthy or pathological). In this paper, it is formulated as a specific instance of variable selection. The originality is that the proteins are not investigated one after the other but the best partition between discriminant and non-discriminant proteins is directly sought. In this way, correlations between the proteins are intrinsically taken into account in the decision. The developed strategy is derived in a Bayesian setting, and the decision is optimal in the sense that it minimizes a global mean error. It is finally based on the posterior probabilities of the partitions. The main difficulty is to calculate these probabilities since they are based on the so-called evidence that require marginalization of all the unknown model parameters. Two models are presented that relate the status to the protein concentrations, depending whether the latter are biomarkers or not. The first model accounts for biological variabilities by assuming that the concentrations are Gaussian distributed with a mean and a covariance matrix that depend on the status only for the biomarkers. The second one is an extension that also takes into account the technical variabilities that may significantly impact the observed concentrations. The main contributions of the paper are: (1) a new Bayesian formulation of the biomarker selection problem, (2) the closed-form expression of the posterior probabilities in the noiseless case, and (3) a suitable approximated solution in the noisy case. The methods are numerically assessed and compared to the state-of-the-art methods (t test, LASSO, Battacharyya distance, FOHSIC) on synthetic and real data from proteins quantified in human serum by mass spectrometry in selected reaction monitoring mode.

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two-dimensional difference gel electrophoresis (2D-DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione-S-transferase Pi, α-enolase, T-complex protein 1 subunit β, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5-15%) and sensitive (0.3 ng·mL(-1)) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver-operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three-marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19-9 reached 0.77. Serum HSP60 appeared to be more specific for late-stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection.

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low microg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 microl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.

International audience; Ribosomal proteins (r-proteins) are increasingly used as an alternative to ribosomal rRNA for prokaryotic systematics. However, their routine use is difficult because r-proteins are often not or wrongly annotated in complete genome sequences, and there is currently no dedicated exhaustive database of r-proteins. RiboDB aims at fulfilling this gap. This weekly updated comprehensive database allows the fast and easy retrieval of r-protein sequences from publicly available complete prokaryotic genome sequences. The current version of RiboDB contains 90 r-proteins from 3,750 prokaryotic complete genomes encompassing 38 phyla/major classes and 1,759 different species. RiboDB is accessible at http://ribodb.univ-lyon1.fr and through ACNUC interfaces. Modern prokaryotic systematics relies mainly on the analysis of the RNA component of the small subunit of the ribosome (SSU-rRNA) and small sets of housekeeping genes (Ludwig and Klenk 2001). The resulting phylogenies have provided interesting but partial information on the evolutionary history of prokaryotes because the corresponding genes do not contain enough phylogenetic signal to resolve with confidence all the species relationships, especially at large and small evolutionary scales. The rise of genomics opens the way for the definition of a novel "gold standard" for prokaryotic systematics that may complement and eventually replace SSU-rRNA. In this respect , ribosomal proteins (r-proteins) were shown to be good candidates (Yutin et al. 2012; Ramulu et al. 2014) because they carry a phylogenetic signal that is consistent with that of larger gene sets such as core genes, while allowing tree inference in acceptable computation time. Moreover, r-proteins are believed to be the main informative molecules detected by MALDI-TOF mass spectrometry, a disruptive technology adopted recently for microbial identification in clinical laboratories (Welker and Moore 2011). The use of r-proteins for phylogenetics and systematics requires the accurate and exhaustive identification of r-proteins homo-logues. However, due to their small size and atypical amino acid composition, r-proteins are often not or badly annotated in genomic sequences (Yutin et al. 2012; Ramulu et al. 2014). This hampers their routine use for systematic purposes. In fact, despite a few attempts in the past 10 years (Nakao et al. 2004; Teeling and Gloeckner 2006), there is currently no up-to-date dedicated database of prokaryotic r-proteins. Here, we present RiboDB, a comprehensive database of pro-karyotic r-proteins built from the reannotation of complete genome sequences available in GenBank. RiboDB gathers sequences of all currently recognized r-protein families (90 r-families), with the exception of S1. In fact, the presence of many repeats of the S1 domain in this r-protein, a conserved domain occurring in a wide range of RNA associated proteins, prevents the reliable identification of S1 homologues. Homologues of each r-family are identified through a double approach combining reciprocal best-blast-hits (rBBH) and hidden Markov model (HMM) profiles (see Supplementary Materials online for details). A regularly updated knowledge database of manually curated r-proteins is used to select the most accurate seeds for rBBH searches and to build the HMM profiles. A scoring system is used to evaluate the accuracy of predicted r-proteins. This double approach was shown to generate very few false positives and false negatives. RiboDB relies on the ACNUC database system (Gouy and Delmotte 2008) and can be accessed through ACNUC interfaces , including the web-based WWW-Query available at http://doua.prabi.fr/search/query_fam. A user-friendly web-site is also available at http://ribodb.univ-lyon1.fr. Both allow the retrieval of r-protein sequences (both amino acids and nucleotides) for user-defined set of taxa and r-families in various formats (e.g., GenBank, fasta). Three output files are Brief communication

Background Cardiac troponin I (cTnI) is a specific marker of myocardial injury. In blood of patients with cardiovascular diseases, cTnI is released as a mixture of free, complexed and post-translationally modified forms. Methods The cTnI forms present in the plasma from 8 patients with acute myocardial infarction (AMI) have been analysed by two-dimensional gel electrophoresis (2-DE) and Western Blot using anti-cTnI mAb 19C7 and anti-phosphorylated cTnI (Serines 22–23) mAb 5E6. Results After immunoextraction of cTnI in plasma samples by 19C7 and 2-DE separation, 4 different forms were detected by 19C7 in 7 out the 8 AMI plasma samples. Two 29 kDa spots corresponding to intact free cTnI forms were detected at pIs 5.2 and 5.4. However, spot with pI 5.4 was also recognized by mAb 5E6, and should be bis-phosphorylated cTnI. Two 55 kDa spots with pIs 6.6 and 6.7 could be IC complexes. CTnI forms with pIs lower than the theoretical pI were also found in free cTnI and phosphorylated cTnI purified materials. Conclusions 2-DE analysis of AMI plasma showed the presence of acidic cTnI forms, one of them being phosphorylated. The clinical significance of these forms has to be further investigated.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an identification procedure for bacteria and fungi. The MALDI-TOF MS-based analysis of resistance to β-lactam antibiotics has been applied to detect hydrolysis of carbapenems by different bacterial strains. However, the detection of enzymatic carbapenem degradation by MALDI-TOF MS lacks well-standardized protocols and several methods and models of interpretation using different calculations of ratio-of-peak intensities have been described in the literature. Here, we used faropenem and ertapenem hydrolysis as model compounds. In an attempt to propose a universal protocol, the hydrolysis was regularly monitored during 24 h using well-characterized bacterial strains producing different types of carbapenemases (KPC, IMP, NDM, VIM, and OXA-48). Variable responses and different timing for detectable hydrolysis, depending on the enzyme produced, were observed. KPC degrades its template antibiotics very quickly (15 min for some KPC producers) compared to other types of enzymes (more than 90 min for other enzymes). Prior bacterial lysis was shown to be of no interest in the modulation or optimization of the hydrolytic kinetics. The adequate detection of carbapenem hydrolysis would, therefore, require several MALDI-TOF MS readouts for the timely detection of rapid hydrolysis without missing slow hydrolysis. This enzymatic constraint limits the implementation of a standard protocol in routine microbiology laboratories.

International audience; The paper proposes a variable selection method for pro-teomics. It aims at selecting, among a set of proteins, those (named biomarkers) which enable to discriminate between two groups of individuals (healthy and pathological). To this end, data is available for a cohort of individuals: the biological state and a measurement of concentrations for a list of proteins. The proposed approach is based on a Bayesian hierarchical model for the dependencies between biological and instrumental variables. The optimal selection function minimizes the Bayesian risk, that is to say the selected set of variables maximizes the posterior probability. The two main contributions are: (1) we do not impose ad-hoc relationships between the variables such as a logistic regression model and (2) we account for instrumental variability through measurement noise. We are then dealing with indirect observations of a mixture of distributions and it results in intricate probability distributions. A closed-form expression of the posterior distributions cannot be derived. Thus, we discuss several approximations and study the robustness to the noise level. Finally, the method is evaluated both on simulated and clinical data. Index Terms— Model and variable selection, Bayesian approach, biological et technological variability, Gaussian mixture, proteomics.

Prostate specific antigen (PSA) is a protease which is characteristic of the prostate. It is widely used as a serum marker for the early diagnosis of prostate cancer (PCa). Nevertheless, for concentrations between 4 and 10 ng/mL, PSA does not enable PCa to be distinguished from benign diseases, such as benign prostate hyperplasia (BPH). In sera, the use of a ratio between free PSA (PSA uncomplexed with protease inhibitor) and total PSA (free PSA and PSA bound to alpha-1 anti-chymotrypsin) enables the "gray zone" to be reduced, but an important proportion of patients are still wrongly classed. Using two-dimensional electrophoresis, we demonstrated using 52 PCa and 40 BPH well-documented clinical cases that BPH sera show a significantly greater percentage of low-molecular-weight free PSA elements (IwPSA) than PCa sera. In our study, the use of a ratio between IwPSA and standard free PSA enables the correct diagnosis of 100% of PCa and 82.5% of BPH cases as against when 73.1% and 42.5% respectively were correctly diagnozed using the total PSA and the free/total PSA ratio. This important finding may be related to differences in the mechanism secreting PSA from the prostate into the bloodstream. We have shown how a tissue marker may be turned into a powerful tumor marker by events probably unrelated to its expression.

The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs).Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane.Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA.The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.

Prostate-specific antigen (PSA), the main marker for prostate cancer (PCa), is released from the prostate into the blood stream at nanogram level and may increase in PCa and nonmalignant disease such as benign prostate hyperplasia (BPH). More recently, advantage was taken of PSA's ability to bind to protease inhibitors in serum in order to improve discrimination between PCa and BPH, using the free PSA to total PSA ratio. The understanding of this phenomenon at molecular level, which is still unknown, may promise new improvements in the field of diagnostics. For this purpose, we determined the pattern of PSA forms in PCa and BPH sera, using the high resolving power of two-dimensional electrophoresis (2-DE) in conjunction with the high sensitivity of chemiluminescence detection. Serum PSA differs drastically from seminal PSA: apart from complexed forms, serum PSA shows few cleaved forms. Moreover, 2-DE patterns from PCa are relatively homogeneous, whereas patterns from BPH may in some cases present a higher proportion of cleaved forms and in other cases present slightly more basic spots. We therefore demonstrated, for the first time, that an increase in the free to total PSA ratio in BPH cases may be due to cleaved PSA forms (which are enzymatically inactive and unable to bind inhibitors), or possibly related to basic free PSA, which may represent the zymogen forms.

International audience; The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005 +/- 5 Thomson, as expected for the naturally formylated delta toxin, or at 3035 +/- 5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

International audience; The transfer of biomarkers from the discovery field to clinical use is still, despite progress, on a road filled with pitfalls. Since the emergence of proteomics, thousands of putative biomarkers have been published, often with overlapping diagnostic capacities. The strengthening of the robustness of discovery technologies, particularly in mass spectrometry, has been followed by intense discussions on establishing well-defined evaluation procedures for the identified targets to ultimately allow the clinical validation and then the clinical use of some of these biomarkers. Some of the obstacles to the evaluation process have been the lack of the availability of quick and easy-to-develop, easy-to-use, robust, specific and sensitive alternative quantitative methods when immunoaffinity-based tests are unavailable. Multiple reaction monitoring (MRM; also called selected reaction monitoring) is currently proving its capabilities as a complementary or alternative technique to ELISA for large biomarker panel evaluation. Here, we present how MRM(3) can overcome the lack of specificity and sensitivity often encountered by MRM when tracking minor proteins diluted by complex biological matrices.

Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS(3) reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM(3)), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 microL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM(3)'s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 10(6)-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM(3) method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzyme-linked immunosorbent assay (ELISA) tests.

Proteomic approaches including high-resolution 2-DE are providing the tools needed to discover disease-associated biomarkers in complex biological samples. Although 2-DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2-DE in polyacrylamide gels. This strategy is based on the 2-DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2-DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with M(r) comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2-DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.

Many problems concerned with the production and the purification of recombinant proteins must be addressed prior to launching an industrial production process. Among these problems, attention is focused on low-level expression that complicates the purification step and can jeopardise the process. The expression of a membrane protein, rP30, of Toxoplasma gondii in the yeast Schizosaccharomyces pombe led to a secretion of only 0.5 μg ml−1. In order to obtain a sufficient quantity for biochemical characterization and evaluation in vitro diagnostic test development, strategies for both production and purification had to be optimized. First, the influence of four nitrogen sources (three peptones and yeast extract) on the growth rate, but also on the separation between the protein and the components of the fermentation broth was assessed. Second, batch and fed-batch fermentations were compared in terms of final biomass and rP30 concentrations. Third, three different protocols that included fixed and expanded bed ion exchange chromatography were compared for processing a large volume of feedstock. By using the most appropriate strategies, i.e. fed-batch fermentation, capture on EBA cation exchanger and affinity chromatography polishing, a purification factor of 1778 and a yield of 49% were achieved. These performances allowed a 12.5-fold increase for the overall rP30 process productivity.

Background: Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer, and the free PSA/total PSA ratio has been shown to be efficient for distinguishing prostate cancer from benign prostatic hyperplasia. We report here the characterization of seven mouse monoclonal antibodies (mAbs) and the partial localization of two conformational epitopes identified by anti-free PSA mAbs.Methods: The mAbs were studied by competition and sandwich assays, and the epitope localization of the two anti-free PSA mAbs (6C8D8 and 5D3D11) was performed using phage displayed peptide libraries and molecular modeling.Results: The seven mAbs were classified into three groups according to their recognition specificities and their ability to inhibit the enzymatic activity of PSA and the formation of PSA-α1-antichymotrypsin (ACT) complex. Among the anti-free PSA mAb group, 6C8D8 recognized the phage displayed peptide RKLRPHWLHFHPVAV, two parts of which presented similarities with two regions distant on the PSA sequence but joined in the tridimensional structure. mAb 5D3D11 recognized the peptide DTPYPWGWLLDEGYD, which is similar to a PSA region located on the board of the groove containing the PSA enzymatic site. Both epitopes were located in the theoretical ACT binding site described previously. Moreover, these mAbs were able to inhibit the enzymatic activity of PSA.Conclusions: These epitope localizations are in agreement with the ability of both mAbs to inhibit enzymatic activity and ACT fixation. The results presented here could bring information for the generation of clinically relevant PSA assays.

For isolation of low soluble recombinant full-length (amino acids 1-191) core protein of hepatitis C virus (HCV) overexpressed in Escherichia coli, the advantage of combining two electrophoretic techniques, in comparison with chromatographic separation, is demonstrated. The protein extract was first solubilized in agents compatible with electrophoretic separation. Using preparative liquid phase isoelectric focusing (IEF) the protein of interest was first concentrated within a defined acidic pH range. These fractions were then submitted to preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) to isolate the 22 kDa protein. The second-dimensional step allowed the isolation of 2 mg of the purified recombinant HCV core protein (rHCV-C191) from 1.5 g bacterial pellet. This quantity is sufficient to characterize the protein and to perform immunogenicity studies. This procedure of two-dimensional preparative electrophoresis is applicable to a wide range of biological samples and represents an alternative for purification of insoluble proteins.

Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.