Robin Tuytten

Highly efficient peptide separations in proteomics Part 2: Bi-and multidimensional liquid-based separation techniques

Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.

Development of an On-Line SPE-LC–ESI-MS Method for Urinary Nucleosides: Hyphenation of Aprotic Boronic Acid Chromatography with Hydrophilic Interaction LC–ESI-MS

The development of an on-line automated SPE-HPLC--ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside levels. The setup comprises a boronate affinity column as a trapping device, a hydrophilic interaction chromatography (HILIC) separation and information-dependent MS detection modes. The system was optimized using standards and tested on biological samples, detecting a number of modified nucleosides. Other urinary biomarkers could also be analyzed by the system developed: for example, the urinary nucleobases were also available for analysis. A simultaneous creatinine-monitoring experiment was also demonstrated to be viable when utilizing the method, which is of benefit as creatinine is a urinary normalizing factor.

Unbiased plasma proteomics for novel diagnostic biomarkers in cardiovascular disease: identification of quiescin Q6 as a candidate biomarker of acutely decompensated heart failure

AIMS Biochemical marker testing has improved the evaluation and management of patients with cardiovascular diseases over the past decade. Natriuretic peptides (NPs), used in clinical practice to assess cardiac dysfunction, exhibit many limitations, however. We used an unbiased proteomics approach for the discovery of novel diagnostic plasma biomarkers of heart failure (HF). METHODS AND RESULTS A proteomics pipeline adapted for very low-abundant plasma proteins was applied to clinical samples from patients admitted with acute decompensated HF (ADHF). Quiescin Q6 (QSOX1), a protein involved in the formation of disulfide bridges, emerged as the best performing marker for ADHF (with an area under the receiver operator characteristic curve of 0.86, 95% confidence interval: 0.79-0.92), and novel isoforms of NPs were also identified. Diagnostic performance of QSOX1 for ADHF was confirmed in 267 prospectively collected subjects of whom 76 had ADHF. Combining QSOX1 to B-type NP (BNP) significantly improved diagnostic accuracy for ADHF by particularly improving specificity. Using thoracic aortic constriction in rats, QSOX1 was specifically induced within both left atria and ventricles at the time of HF onset. CONCLUSION The novel biomarker QSOX1 accurately identifies ADHF, particularly when combined with BNP. Through both clinical and experimental studies we provide lines of evidence for a link between ADHF and cardiovascular production of QSOX1.

Integrated proteomics pipeline yields novel biomarkers for predicting preeclampsia

Preeclampsia, a hypertensive pregnancy complication, is largely unpredictable in healthy nulliparous pregnant women. Accurate preeclampsia prediction in this population would transform antenatal care. To identify novel protein markers relevant to the prediction of preeclampsia, a 3-step mass spectrometric work flow was applied. On selection of candidate biomarkers, mostly from an unbiased discovery experiment (19 women), targeted quantitation was used to verify and validate candidate biomarkers in 2 independent cohorts from the SCOPE (SCreening fOr Pregnancy Endpoints) study. Candidate proteins were measured in plasma specimens collected at 19 to 21 weeks’ gestation from 100 women who later developed preeclampsia and 200 women without preeclampsia recruited from Australia and New Zealand. Protein levels (n=25), age, and blood pressure were then analyzed using logistic regression to identify multimarker models (maximum 6 markers) that met predefined criteria: sensitivity ≥50% at 20% positive predictive value. These 44 algorithms were then tested in an independent European cohort (n=300) yielding 8 validated models. These 8 models detected 50% to 56% of preeclampsia cases in the training and validation sets; the detection rate for preterm preeclampsia cases was 80%. Validated models combine insulin-like growth factor acid labile subunit and soluble endoglin, supplemented with maximally 4 markers of placental growth factor, serine peptidase inhibitor Kunitz type 1, melanoma cell adhesion molecule, selenoprotein P, and blood pressure. Predictive performances were maintained when exchanging mass spectrometry measurements with ELISA measurements for insulin-like growth factor acid labile subunit. In conclusion, we demonstrated that biomarker combinations centered on insulin-like growth factor acid labile subunit have the potential to predict preeclampsia in healthy nulliparous women.

In-Source CID of Guanosine: Gas Phase Ion-Molecule Reactions

In-source collision induced dissociation was applied to access second generation ions of protonated guanosine. The in-source gas-phase behavior of [BH2]+-NH3 (m/z 135, C5H3N4O+) was investigated. Adduct formation and reactions with available solvent molecules (H2O and CH3OH) were demonstrated. Several addition/elimination sequences were observed for this particular ion and solvent molecules. Dissociation pathways for the newly formed ions were developed using a QqTOF mass spectrometer, permitting the assignment of elemental compositions of all product ions produced. Reaction schemes were suggested arising from the ring-opened intermediate of the protonated base moiety [BH2]+, obtained from fragmentation of guanosine. The mass spectral data revealed that the in-source CH3OH-reaction product underwent more complex fragmentations than the comparable ion following reaction with H2O. A rearrangement and a parallel radical dissociation pathway were discerned. Apart from the mass spectrometric evidence, the fragmentation schemes are supported by density functional theory calculations, in which the reaction of the ring-opened protonated guanine intermediate with CH3OH and a number of subsequent fragmentations were elaborated. Additionally, an in-source transition from the ring-opened intermediate of protonated guanine to the ring-opened intermediate of protonated xanthine was suggested. For comparison, a low-energy collision induced dissociation study of xanthosine was performed. Its dissociation pathways agreed with our assumption.

Toward clinical proteomics on a next-generation sequencing platform.

We report the first next generation sequencing (NGS) application to identify and quantify proteins. Customization of protein specific aptamers enabled direct conversion of serum protein information into NGS read outs. The intrinsic ability of aptamer sequencing to highly multiplex protein detection and quantification, together with the prospect of DNA sequencing further evolving into a commodity technology, could constitute the core of a novel, universal diagnostics paradigm.

Mid-trimester maternal ADAM12 levels differ according to fetal gender in pregnancies complicated by preeclampsia

An overrepresentation of adverse pregnancy outcomes has been observed in pregnancies associated with a male fetus. We investigated the association between fetal gender and candidate biomarkers for preeclampsia. Proteins were quantified in samples taken at 20 weeks from women recruited to the SCreening fOr Pregnancy Endpoints (SCOPE) study (preeclampsia n = 150; no preeclampsia n = 450). In contrast to placental growth factor, soluble endoglin, and insulin-like growth factor acid labile subunit, levels of metallopeptidase domain 12 (ADAM12) at 20 weeks were dependent on fetal gender in pregnancies complicated by preeclampsia, for male (n = 73) fetuses the multiples of the median (MoM; interquartile range [IQR] 1.1-1.5) was 1.3, whereas for female fetuses (n = 75) MoM was 1.1 (1.0-1.3); P < .01. Prediction of preeclampsia using ADAM12 levels was improved for pregnancies associated with a male fetus (area under receiver–operator curve [AUC] 0.73 [95% confidence interval [CI] 0.67-0.80]) than that of a female fetus (AUC 0.62 [0.55-0.70]); P = .03. The data presented here fit a contemporary hypothesis that there is a difference between the genders in response to an adverse maternal environment and suggest that an alteration in ADAM12 may reflect an altered placental response in pregnancies subsequently complicated by preeclampsia.

Application of a combined weak cation-exchange/crown ether column: first demonstrations of a versatile tool for proteome subselection.

The present paper introduces the use of a weak cation-exchange/crown ether column in the proteomics field. The 18-crown-6 ether functionality is well-known to selectively complex ammonium and monoalkylammonium ions, which should make this column highly suitable to trap peptides with free alpha-NH(2) or free epsilon-NH(2) groups from lysine side chains. This unique selection mechanism was put to the test in an N-teromics setup which aims for the enrichment of deliberately acetylated protein N-terminal peptides from a serum digest. It was demonstrated that peptides with free alpha-NH(2) groups and peptides with alpha-amino-acetylated groups can be separated from each other using this weak cation-exchange/crown ether column. The peptides of interest, bearing no free primary amines, were found to be significantly enriched in the columns flow through. At the same time a favorable coenrichment of N-glycosylated peptides was observed. To obtain more insight in the contributions of the two distinct column functionalities, i.e., the weak cation exchanger and the crown ether, the experimental data were checked against a theoretical prediction of the outcome.

Revealing of endogenous Marinobufagin by an ultra-specific and sensitive UHPLC-MS/MS assay in pregnant women

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

D3. Early prediction of preeclampsia risk assessment: analytical determination for marinobufagenin in pregnant women

Abstract Introduction: Marinobufagenin (MBG), an endogenous bufadienolide cardiac inotrope, is responsible for vasoconstriction and natriuresis [1,2]. Growing interest by its implication in volume expansion mediated hypertensive states such as essential hypertension, congestive heart failure, chronic renal failure, primary aldosterism and preeclampsia (PE). Elevated endogenous MBG levels have been described early in preeclamptic patients [2–5]. Aims: To develop a specific and sensitive analytical MBG assay with special attention for the limit of quantification (LOQ). An algorithm dealing with the MBG plasma levels might be established in the future, in order to help for prediction of the risk for preeclampsia in pregnant women. Method: A LC-MS/MS based assay designed to determine MBG in human plasma is being optimized. The obtained LOQ fully satisfies the need for quantification of MBG plasma levels in pregnancy (nmol/L range). After a validation process, a primary observational clinical study will allow us to confirm previous results observed in preeclampsia. Results and discussion: We had the opportunity to authenticate the presence of MBG by LC-MS/MS in non-pregnant and in early-pregnant women. These pioneering preliminary results that are giving a promising perspective for early preeclampsia risk assessment in pregnant women will be confirmed with the clinical study.