bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/352468

Validation and automation of a high-throughput multi-targeted method for semi-quantification of endogenous metabolites from different biological matrices using tandem mass spectrometry

Nandania, J.·Peddinti, G.·Pessia, A.·Kokkonen, M.·Velagapudi, V.
View original source ↗Export metadata ↓

Abstract

The use of metabolomics profiling to understand metabolism under different physiological states has increased in recent years, which created the need for robust analytical platforms. Here, we present a validated method for targeted and semi-quantitative analysis of 102 polar metabolites that covers major metabolic pathways from 24 classes in a single 17.5-min assay. The method has been optimized for a wide range of biological matrices from various organisms, and involves automated sample preparation, and data processing using in-house developed R package. To ensure reliability, the method was validated for accuracy, precision, selectivity, specificity, linearity, recovery, and stability according to European Medicines Agency guidelines. We demonstrated excellent repeatability of the retention times (CV<4%), calibration curves (R2[&ge;]0.980) in their respective wide dynamic concentration ranges (CV<3%), and concentrations (CV<25%) of quality control samples interspersed within 25 batches analyzed over a period of one-year. The robustness was demonstrated through high correlation between metabolite concentrations measured using our method and NIST reference values (R2=0.967), including cross-platform comparability against the BIOCRATES AbsoluteIDQp180 kit (R2=0.975) and NMR analyses (R2=0.884). We have shown that our method can be successfully applied in many biomedical research fields and clinical trials, including epidemiological studies for biomarker discovery. In summary, a thorough validation demonstrated that our method is reproducible, robust, reliable, and suitable for metabolomics studies.

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Nandania, J., Peddinti, G., Pessia, A., Kokkonen, M., Velagapudi, V.. 2018-06-20. Validation and automation of a high-throughput multi-targeted method for semi-quantification of endogenous metabolites from different biological matrices using tandem mass spectrometry. https://doi.org/10.1101/352468

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Functional Reconstitution of Human OATP1B1 and OATP1B3 in Proteoliposomes: Effect of Bicarbonate Gradients and Substrate-specific Stimulation on Transport

The hepatic organic anion transporting polypeptides OATP1B1 and OATP1B3 are critical for the clearance of widely prescribed drugs, hepatotoxins, and endogenous metabolites. However, the thermodynamic driving force governing their transport has remained a subject of debate due to the complexity of cellular models. Here, we report a defined, reconstituted proteoliposome system to study the transport kinetics of isolated human OATP1B1 and OATP1B3. Through direct functional assays, real-time counter-ion tracking, and a novel ABCG2-OATP1B1 functional co-reconstitution, we identify an outwardly directed bicarbonate gradient as a thermodynamic driving force for concentrative substrate uptake. In contrast we find no evidence for the proposed role of reduced glutathione efflux as a possible driving force. Targeted mutagenesis experiments revealed that the conserved TM3 residue His115 is needed to couple the bicarbonate gradient to concentrative substrate uptake, mechanistically linking substrate antiport to local pH-sensitive bicarbonate processing. We also investigated the substrate-specific stimulatory effect of the green tea catechin epigallocatechin gallate (EGCG) on OATP1B3 transport, confirming that this effect is intrinsic to OATP1B3, substrate specific, and does not need the involvement of other cellular proteins. Our findings define the energetic basis of OATP1B1 and OATP1B3 hepatic xenobiotic clearance.

biochemistry↗
bioRxiv2026

Mapping the sequence determinants of constitutive activity in GPCRs

G protein-coupled receptors (GPCRs) signal not only in response to ligands but also in their absence. This basal, or constitutive, activity has distinct physiological consequences and is the only functional measurement available for orphan receptors. It is also hard to compare receptors because each is reported relative to its own baseline. Here, we measure basal activity on a single scale across the human GPCR family. Pairing a barcoded library of all human GPCRs with a transcriptional cAMP reporter, we scored 901 receptor constructs in one pool experiment. Basal activity forms a continuous distribution, and many of the highest-scoring recipes have prior reports of ligand-independent activity, with orphan being strongly represented. We then exhaustively mutated one of them, GPR161, and measured its effects on protein abundance, surface expression, and cAMP signaling. Of the 528 positions, only 10 specifically alter signaling independent of expression. Constraints on basal signaling are dominated by reepotro biogenesis, which requires multiphenotype scanning to resolve. Together, these datasets place basal activity on a common scale across the GPCR family and provide a template for dissecting it at residue resolution.

biochemistry↗
bioRxiv2026

Direct Bioconjugations of Diverse Payloads using an Extraordinarily Donor-Promiscuous Flavin Transferase

Site-specific protein conjugation is a widely used tool in chemical biology research and is the foundation of many biotechnological and biomedical applications. A key enabling technology are repurposed post-translational protein modifying enzymes, where variants with broad donor and acceptor substrate promiscuity were developed into general-purpose bioconjugation platforms. However, direct enzymatic attachment of bulky or structurally diverse payloads is often limited by low reaction efficiencies and difficulties in preparing suitable donors. Consequently, conjugation typically relies on two-step strategies in which an enzyme first installs a bioorthogonal handle on the target protein, followed by chemical attachment of the payload. These enzyme-first, chemistry-second approaches limit chemistry to bioorthogonal reactions that leave a molecular footprint. Here, we introduce direct ADDing, an enzymatic bioconjugation platform based on our discovery of the exceptional donor promiscuity of the flavin transferase ApbE. We show that ApbE accepts a remarkably broad range of donor substrates provided they carry an adenosine diphosphate (ADP) anchor. The prevalence of ADP-containing compounds in nature provides access to diverse building blocks and an extensive biochemical toolbox for donor generation. Exploiting these resources, we demonstrate multiple strategies for preparing compatible donors and achieve direct, site-specific enzymatic conjugation of diverse small molecules, peptides, proteins, and synthetic polymers.

biochemistry↗