OS043. Identification and validation of novel markers for the predictionof pre-eclampsia.

INTRODUCTION: Currently no test accurately predicts pre-eclampsia (PE) in a healthy nulliparous population. Unbiased protein biomarker discovery has the potential to identify novel markers but multimarker panels are required to achieve clinically relevant prediction of PE. To this purpose, single biomarker performances were obtained and multimarker panels developed in a significant subcohort of the international Screening fOr Pregnancy Endpoints study (SCOPE) study [1]. OBJECTIVES: To identify and validate novel protein markers for PE prediction using chromatographic and mass spectrometric techniques which enable the identification and quantification of plasma proteins present in plasma at sub ng/ml concentration (Pronota, Belgium). METHODS: Pre-disease plasma samples (22-26 weeks) from women who subsequently developed PE and those with uncomplicated pregnancies [2] were used to generate 30 plasma proteome profiles using the MASStermind™ pipeline. A set of novel protein candidates were validated using an antibody-free mass spectrometry method using multiple reaction monitoring (MASSterclass™) in a subcohort of the SCOPE study (NZ & Aus) [1]. Relative abundance of 40+ proteins was determined in 20week plasma samples from 100 women who developed PE and 200 women who did not develop PE (included women with other pregnancy complications). Multivariate analyses were performed to identify algorithms with predictive performance using combinations confined to a maximum of 6 parameters (protein markers and clinical parameters) to avoid overfitting. Validation of the prediction panels was performed in an independent subcohort of SCOPE (Europe) comprising 50 PE and 150 no PE. RESULTS: From this large scale biomarker discovery effort a number of key results were obtained: a novel protein, i.e., Insulin-like growth factor binding protein, acid labile subunit (IGFALS), was identified. AUC for this marker for the prediction of all PE was 0.71 (CI 0.68-0.75) which was greater than both PlGF and s-Eng (respective AUCs: 0.64 and 0.61). IGFALS was also found to have predictive value for term (AUC 0.70) as well as preterm disease (AUC 0.75). Using multivariate analysis, marker panels were identified that achieved clinically relevant prediction (exemplary panel prediction of all PE cases AUC=0.79; prediction of preterm PE AUC=0.92). These multivariate models were successfully validated in the European SCOPE subcohort. In addition, predictive algorithms based on mass spectrometric read outs were largely invariant to interchanging the IGFALS mass spectrometry quantitation data with IGFALS ELISA data. CONCLUSION: This study demonstrates the capability of high level LC-MS technologies to discover candidate biomarkers and execute large scale multiplex validation to develop a predictive screening test for preeclampsia.

Online immobilized metal affinity chromatography/mass spectrometric analysis of changes elicited by cCMP in the murine brain phosphoproteome.

An automated online immobilized metal affinity chromatography/high-performance liquid chromatography mass spectrometric (IMAC-HPLC/MS/MS) method was developed to study cytidine 3',5'-cyclic monophosphate (cCMP)-specific protein phosphorylation, analogous to a previously successful offline IMAC method using microvolume IMAC pipette tips. The optimized method identified murine brain phosphoproteins selectively modified by challenge with cCMP, using manual interpretation of the results to confirm both phosphorylation and selectivity of response to cCMP. A number of proteins identified by this strategy have potential roles in hyperproliferation, a previously reported response to elevated levels of cCMP.

Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?

A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.

Short capillary ion-pair high-performance liquid chromatography coupled to electrospray (tandem) mass spectrometry for the simultaneous analysis of nucleoside mono-, di- and triphosphates.

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.