CIL-ExPMRM: An Ultrasensitive Chemical Isotope Labeling Assisted Pseudo-MRM Platform to Accelerate Exposomic Suspect Screening.

Exposome is the future of next-generation environmental health to establish the association between environmental exposure and diseases. However, due to low concentrations of exposure chemicals, exposome has been hampered by lacking an effective analytical platform to characterize its composition. In this study, by combining the benefit of chemical isotope labeling and pseudo-multiple reaction monitoring (CIL-pseudo-MRM), we have developed one highly sensitive and high-throughput platform (CIL-ExPMRM) by isotope labeling urinary exposure biomarkers. Dansyl chloride (DnsCl), N-methylphenylethylamine (MPEA), and their isotope-labeled forms were used to derivatize polar hydroxyl and carboxyl compounds, respectively. We have programmed a series of scripts to optimize MRM transition parameters, curate the MRM database (>70,000 compounds), predict accurate retention time (RT), and automize dynamic MRMs. This was followed by an automated MRM peak assignment, peak alignment, and statistical analysis. A computational pipeline was eventually incorporated into a user-friendly website interface, named CIL-ExPMRM (http://www.exposomemrm.com/). The performance of this platform has been validated with a relatively low false positive rate (10.7%) across instrumental platforms. CIL-ExPMRM has systematically overcome key bottlenecks of exposome studies to some extent and outperforms previous methods due to its independence of MS/MS availability, accurate RT prediction, and collision energy optimization, as well as the ultrasensitivity and automated robust intensity-based quantification. Overall, CIL-ExPMRM has great potential to advance the exposomic studies based on urinary biomarkers.

Detection and differentiation of active and inactive isoforms of coagulation factors II, VII, IX, and X in prothrombin complex concentrate by mass spectrometry.

PURPOSE: Prothrombin complex concentrates (PCCs) are plasma products containing a mixture of four inactive/proactive coagulation factors. The activated forms of human coagulation factors, like Thrombin (FIIa), Convertin (FVIIa), activated Christmas factor (FIXa) and the activated Stuart-Prower factor (FXa), are impurities in PCCs. Until now no valid assay exists to differentiate the non activated proform (inactive) from active coagulation factor isoforms in PCCs in one measurement. Therefore, the aim of this study was to establish a mass spectrometry (LC-MS/MS)-based assay to address this issue in the ready to use medicinal product. METHODS: Bottom-up proteomics combining double digestion (Glu-C & Lys-C) and LC-MS/MS, was used to differentiate the inactive and active forms of the coagulation factors Prothrombin (FII), Proconvertin (FVII), Christmas factor (FIX) and the Stuart-Prower-factor (FX) in PCCs. RESULTS AND CONCLUSIONS: A targeted pseudo-multiple reaction monitoring (pMRM-LC-MS/MS)-assay was developed for the specific detection of four different coagulation factors in PCCs. Proteotypic peptides for the inactive/active isoforms (zymogen) of the four coagulation factors were identified and validated by the investigation of six investigational and one commercially available PCCs. In conclusion, the semi-quantitative determination and the distinction between the active and the inactive isoform of the respective coagulation factors were possible in one liquid chromatography tandem mass spectrometry (LC-MS/MS) run.

Bioanalysis of plasma acetate levels without derivatization by LC-MS/MS.

Background: The acetate ion has important physiological functions and important therapeutic applications. A rapid LC-MS/MS method is described to measure acetate ions in human plasma without chemical derivatization. Materials & methods: A 200 µl sample was spiked with the internal standard 1,2-13C-acetate and proteins precipitated with trichloroacetic acid. The supernatant was recovered and separated under acidic conditions on a C18-column. The eluent was alkalinized by post-column infusion of methanolic ammonium hydroxide. Acetate ions were monitored on a low resolution mass spectrometer in negative ion mode. Results: Method was validated for accuracy and precision with a lower limit of quantitation of 9.7 µM and linear dynamic range up to 339.6 µM. Conclusion: The method is open for analytical improvement and adapts with metabolomic and pharmacometabolomic studies on chemicals of similar nature.

A novel LC-MS/MS method for the quantitative measurement of the acetate content in pharmaceutical peptides.

Most pharmaceutical peptides are supplied as acetate salts and the relative amount of acetate to peptide in the final product is one quality criterion required by regulatory agencies to approve the product for clinical use. The objective of the present study was to develop a validated LC-MS/MS method that allows the quantitative determination of the acetate content in pharmaceutical peptide preparations and simultaneous monitoring and collection of qualitative and quantitative information on the peptide during manufacture and in the final product. The method uses reversed phase C18-chromatography to elute the acetate ions under acidic conditions, pH 3, followed by post-column infusion of ammonium hydroxide 0.6M, methanolic solution (30:70) at a rate of 0.5mL/hr. The acetate ions were monitored in negative polarity mass spectrometry by pseudo multiple reaction monitoring (pseudo MRM) against 1, 2- 13C labelled acetate, the internal standard used in the method. The method was linear for acetate concentrations between 0.4 and 25µg/mL with a coefficient of determination (r2) equal to 0.9999. The minimum level of detection and minimum level of quantification were at 0.06µg/mL and 0.18µg/mL respectively. Accuracy of the method was judged by determining the acetate content in a commercial product of the peptide pharmaceutical tetracosactide (TCS) and parallel comparison to the amounts determined by a reversed phase HPLC method with detection at a wavelength of 210nm. The amounts determined by the two methods were in agreement with a RSD that was less than 2%. Additional confirmation of method accuracy was determined by spiking the pharmaceutical peptide with varying amounts of acetate. The recoveries ranged on average between 101 and 102% for the spiked amounts. Accuracy was also determined by calculating the percentage relative error of the predicted to actual acetate concentration in quality controls and was determined to be less than 5%. The LC-MS/MS method was precise with an intra- and inter-day RSD of less than 5%. The standard solutions were stable for at least one month when kept frozen at -80°C with no loss in response and an inter-day RSD of less than 5%. The method was applied to quantify the acetate content in the clinically available product of TCS and to simultaneously evaluate the average peptide molecular weight and detect known impurities by switching from negative polarity MRM analysis to positive polarity MS analysis following the elution of the acetate peak. The method reported herein should corroborate quantitative determinations of the acetate content in pharmaceuticals by the traditional compendial HPLC method.

Development and characterization of a pseudo multiple reaction monitoring method for the quantification of human uromodulin in urine.

BACKGROUND: Uromodulin is the most abundant protein in healthy human urine. Recently it has been suggested as a specific biomarker of renal tubular damage. We have developed a novel pseudo multiple reaction monitoring (pseudo MRM) for the protein's quantification in human urine. RESULTS: Selection of two peptides allowed quantification of uromodulin in human urine. The pseudo MRM quantified uromodulin in healthy individuals between 21 and 1344 nM and in autosomal dominant tubulointerstitial kidney disease-UMOD patients between 2 and 25 nM. CONCLUSION: The pseudo MRM allows greater confidence in assay specificity than traditional MRM methods and quantified uromodulin at concentrations higher than achievable by ELISA. Differences in urinary uromodulin concentration related to the rs4293393 promoter variant in the UMOD gene was confirmed. This method will be used to further investigate uromodulin as a biomarker of renal injury.

Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry.

Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.