Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., β-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020.

Determination of potential mobility of toxic trace metals in sediments under changing redox condition is important in ecological risk assessment. Current methods are limited in risk prediction in such dynamic environment. In this study, we have discussed the general disagreement from widely used methods (sediment quality guideline (SQGs), potential ecological risk index (PERI), risk assessment code (RAC) using BCR fraction information). In addition, the stable isotopic dilution method (IDM) was also modified to quantify metal lability in a microcosm experiment mimicking river bank sediment turning into anaerobic. The isotopically exchangeable Cd, Cu, Pb, and Zn quantified by IDM (%E incub) was used in the RAC to reveal the trend of risk during this process. Strong risks from Cd are suggested by the PERI and RAC as a result of high toxicity and mobility of the element, while SQGs suggests medium risk for Cu, Pb, and Zn in certain samples. The disagreement between the results of RAC assessed by metal lability (%E dry) and by BCR metal fractionation reflects the effect of sediment properties and source of metal contamination. The RAC based on the non-residual fractions is likely to overestimate the potential risk for most metals even there is a significant change in sediment Eh. The RAC assessed by %E incub reveals that the variability in risk in response to the reducing Eh is not consistent. Large fluctuation in %E incub for Cd (28.5%, 49.5%), Pb (27.6%, 18.2%), and Cu (14.4%, 24.7%) can shift the risks to a higher level in certain range of Eh in two sediments. In sediment with lower contents of metal binding phases (e.g. mineral oxides, organic matters), the release of metals can be more significant, thus higher ecological risk in changing redox condition.

The purpose of this work was to compare the measured red-cell volume (RCV) using sodium pertechnétate [RCV-99mTc] compared to the reference technique using sodium radiochromate [RCV-51Cr] and to assess the influence of technetium-99 elution on the RCV-99mTc value. Ten patients had simultaneous measurements of RCV-99mTc and RCV-51Cr. Elution of Tc-99m from red blood cells was 2.9% and led to an average overestimation of RCV-99mTc of 3.7%. The introduction of individual tracer elution rates in the RCV-99mTc calculation corrects this overestimation.

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N6-Formyl-lysine (FLys) is an abundant and lasting protein adduct formed when formaldehyde generated by nitrosative/oxidative stress and inflammation reacts with lysine residues. It is believed that the post-translational N6-formylation of lysine is associated with a variety of pathological processes and human diseases. Thus, FLys may serve well as a dosimetric biomarker for exposure to formaldehyde and other oxidative stress-inducing toxicants. However, since current methods for FLys determination are tedious and time-consuming, we developed and validated an aqueous normal phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with isotope-dilution method for the rigorous quantification of FLys with enhanced sensitivity and selectivity. After validating the accuracy and precision of the method with a synthetic peptide containing FLys, the method was applied to quantitate the concentration-dependent formation of FLys in cells exposed to formaldehyde and Fe2+-EDTA, an OH radical-mediated oxidant. The study reveals formaldehyde and Fe2+-EDTA produced FLys at a frequency of 20.2 and 4.1 per 104 lysine per mM, respectively, after correcting for losses during protein digestion steps. The study was further extended to quantitate the concentration-dependent formation of FLys in aristolochic acid I (AA-I) exposed Escherichia coli cells and rat tissues. This study demonstrates for the first time that AA-I exposure induces time- and dose-dependent formation of FLys in cellular proteins. Furthermore, results show AA-I exposure leads to organotropic N6-formylation of lysine, with elevated levels of FLys detectable in the kidney, which is the one of the tumor targeting organs of AAs. Previous studies have also revealed AA exposure induced renal interstitial fibrosis in both laboratory rodents and humans, by a yet to be determined molecular mechanism. These data shed light on the potential caustative role of N6-formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.

Volume management is an essential component of anti-hypertensive therapy. Different volume phenotypes have been proposed. We sought to study the total blood volume (TBV), plasma volume (PV), and red blood cell volume (RBV) in hypertensive patients. We included patients followed in an outpatient cardiology clinic from 1998 to 2003. Blood volume (BV) parameters were measured using radioisotope iodine-131-labeled albumin dilution technique. Values were expressed as percentage (%) deviation from ideal volumes. A total of 95 patients were included. The intravascular volume distribution as percent deviation from normal volume ranged from - 23 to + 28% for TBV, - 22 to + 36% for PV and - 29 to + 37% for RBV. There was no significant correlation between systolic BP and any of the BV parameters (TBV and SBP, r = - 0.03; PV and SBP, r = - 0.12; RBV and SBP, r = - 0.08). Patients with hypertension have a wide variation in BV parameters. BV does not correlate with SBP.

Caenorhabditis elegans is used extensively as a medical and toxicological model organism. However, little is known about background levels of oxidatively induced DNA damage in the nematode or how culturing methods affect DNA damage levels. The tough C. elegans cuticle makes it challenging to extract genomic DNA without harsh procedures that can artifactually increase DNA damage. Therefore, a mild extraction protocol based on enzymatic digestion of the C. elegans cuticle with high-salt phase-separation of DNA has been developed and optimized. This method allows for efficient extraction of >50 μg DNA using a minimum of 250000 nematodes grown in liquid culture. The extracted DNA exhibited acceptable RNA levels (<10% contamination), functionality in polymerase chain reaction assays, and reproducible DNA fragmentation. Gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution measured lower lesion levels in high-salt extracts than in phenol extracts. Phenolic extraction produced a statistically significant increase in 8-hydroxyguanine, a known artifact, and additional artifactual increases in 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine, and 8-hydroxyadenine. The high-salt DNA extraction procedure utilizes green solvents and reagents and minimizes artifactual DNA damage, making it more suitable for molecular and toxicological studies in C. elegans. This is, to our knowledge, the first use of GC-MS/MS to measure multiple 8,5\'-cyclopurine-2\'-deoxynucleosides in a toxicologically important terrestrial organism.

The use of isotope-labeled internal standards is the most widely accepted approach to overcome the matrix effects on quantification of pesticides in food by LC/MS. We evaluated the impact of the matrix effects on quantification of six neonicotinoid pesticides, acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam, in food by using deuterated internal standards. The calibration curves for each pesticide were obtained by using matrix-free and matrix-matched calibration solutions with blank brown rice, carrot, and green onion extracts. For brown rice and carrot, the matrix effects were not observed. In contrast, the slopes of calibration curves for each pesticide were influenced by presence of green onion extracts in calibration solutions (variability of the slopes was 4-9%), because the ratios of peak area for native pesticide to those for internal standards were influenced by matrix. The spike-and-recovery test with green onion was also performed. The analytical values obtained by using matrix-free calibration solution were biased from the spiked concentration, whereas those obtained by using matrix-matched calibration solution were comparable to the spiked concentration. These results indicate that matrix-matched calibration solution should be used for accurate quantification of neonicotinoid pesticides in food by LC/MS using deuterated internal standards.

Microcystins are cyclic peptide toxins with hepatotoxic and tumor-promoting properties, which are produced in significant quantities (up to tens of μg/L) in freshwater cyanobacterial water blooms. Several studies reported microcystin accumulation in fish with possible food transfer to humans. These compounds are further metabolized to cysteine and glutathione conjugates which can be present in tissues in significant concentrations. In this study, we focused on the development and evaluation of robust and highly sensitive SPE-LC-MS/MS method for the analysis of microcystin conjugates in fish tissue samples. For the first time, we demonstrate the use of isotopically labeled internal standards which are essential for accurate and precise determination of analytes in complex biotic matrices. LLOQs of respective microcystin conjugates (signal-to-noise ratio; S/N > 10, peak-to-peak method) ranged from 3.3 to 5.0 ng/g of tissue fresh weight (FW). The calibration was linear within a range of concentrations from 1 to 70 ng/mL for all analyzed conjugates. The precision and repeatability of the method were very good with recoveries in the range of 88.5-107.6% and relative standard deviations between 8.8 and 13.2% for all analytes. In the follow-up study, fully validated method was used for the determination of microcystin conjugate levels in common carp exposed to microcystin-containing cyanobacterial biomass under controlled conditions. Significant amounts of microcystin conjugates (up to 55 ng/g) were found in the tissues of fish after 7 weeks of exposure. Our method was shown to be robust, sensitive, selective, and suitable for the determination of trace levels of microcystin conjugates in fish tissues.