Application Note 38 describes stable isotope dimethyl labelling as a simple, fast, and cost-effective method for quantitative MS-based proteomics. The technique chemically modifies primary amines in peptides using isotopically distinct forms of formaldehyde and cyanoborohydride to generate light, intermediate, and heavy labels that differ in mass, enabling multiplexed sample comparison in a single analysis. Particularly useful for tissues and samples where metabolic labelling (e.g., SILAC) is impractical, dimethyl labelling is compatible with common digestion and fractionation workflows and has been successfully applied to proteome and phosphoproteome studies, including large-scale comparisons of stem cell proteomes.

Application Note 37 presents INLIGHT™ (Individuality Normalization when Labelling with Isotopic Glycan Hydrazide Tags), a strategy for relative quantification of N-linked glycans by LC-MS. The method uses hydrophobic hydrazide reagents (available in light and ¹³C₆-labelled heavy forms) to derivatize PNGase F–released N-glycans via efficient hydrazone formation, improving electrospray ionization response and enabling reversed-phase chromatography. After labelling two samples separately, they are mixed 1:1 and analysed together, with data processing that includes isotopic overlap correction and total glycan normalization to provide accurate relative quantification. The approach enhances sensitivity, minimizes sample loss, and offers a robust workflow for comparative glycomics studies.

Application Note 17 describes the use of adenosine 5′-triphosphate (γ-P¹⁸O₄, 97%) as a non-radioactive method for the unambiguous identification of phosphopeptides by mass spectrometry. In vitro kinase reactions are performed with a 1:1 mixture of normal ATP and ¹⁸O-labelled ATP, producing phosphopeptides that appear as characteristic mass doublets (Δ6.01 Da) or triplets for multiply phosphorylated peptides, providing a clear isotopic signature that confirms phosphorylation status. The study demonstrates that no isotope exchange occurs during reactions and highlights the advantages of this stable isotope approach over traditional γ-³²P methods, including improved safety, easier handling, and reduced false positives in phosphopeptide identification.

Application Note 11 reviews the role of stable isotope labelling in quantitative proteomics, highlighting how mass spectrometry combined with isotopic strategies enables accurate measurement of relative protein abundance and post-translational modifications. The note describes metabolic labelling approaches (e.g., ¹⁵N enrichment or isotope-depleted media), chemical labelling methods such as ICAT for cysteine-containing peptides, and the PhIAT strategy for quantifying phosphorylation. By generating isotopically distinct versions of proteins or peptides that can be analysed simultaneously, these techniques improve quantitation, reduce variability, and expand the capability of MS-based proteome-wide comparisons.

Application Note 45 describes the use of metabolic isotopic analysis (MIA) with ¹³C₆-glucose to investigate mitochondrial function in pRb-deficient cells in vivo. Using isotope tracing in mice and human RPE cell models, the study shows that loss of pRb increases glucose entry into the TCA cycle (elevated M+2 citrate and PDH activity) but paradoxically reduces overall TCA cycle flux and ATP production, indicating impaired oxidative phosphorylation rather than enhanced glycolysis. Additional glutamine tracing and sensitivity to mitochondrial inhibitors confirm diminished mitochondrial capacity, revealing a metabolic vulnerability in pRb-deficient cells relevant to oncology research.

Application Note 43 describes the use of a custom 20% ¹³C₆-leucine and 20% ¹³C₅-valine mouse feed to study whole-body branched-chain amino acid (BCAA) metabolism in mice. Using pulse-feeding and pulse-chase experimental designs combined with GC-MS analysis, the study distinguishes between short-term dietary uptake and long-term contributions from tissue protein turnover to plasma BCAA levels. Results show rapid absorption and disposal of dietary BCAAs and demonstrate that approximately two-thirds of plasma BCAAs in the fed state derive from long-term protein stores. The approach provides a robust framework for investigating systemic BCAA metabolism in cancer, diabetes, and other metabolic diseases.

Application Note 32 describes an in vivo SILAC-based proteomic approach for analysing tyrosine kinase (SRC) signalling in human cancer xenografts using Mouse Express® L-lysine (¹³C₆, 99%) mouse feed. By feeding nude mice a heavy lysine diet for 30 days after implantation of human colorectal cancer cells, researchers achieved >88% tumour proteome labelling, enabling quantitative phosphotyrosine profiling by LC-MS/MS. This approach identified 61 SRC-dependent targets in vivo, many of which differed from those observed in cell culture, demonstrating that oncogenic signalling in tumours significantly diverges from in vitro models and highlighting the value of in vivo stable isotope labelling for studying cancer signalling pathways.

Application Note 27 describes a targeted LC-SRM/MS method for multiplexed quantification of synaptic proteins using Mouse Express® brain tissue labelled with L-lysine (¹³C₆) as a SILAM internal standard. Light synaptic vesicle, presynaptic, and postsynaptic density fractions from mouse brain were mixed with heavy labelled membrane preparations, separated, digested, and analysed by triple quadrupole MS to determine light/heavy peptide ratios. The approach enabled quantification of over 100 synaptic proteins with microdomain specificity, revealing distinct enrichment patterns—such as kinases and scaffold proteins in the postsynaptic density and SNARE proteins in vesicles. This method provides a robust, reproducible alternative to peptide standards or isobaric tagging and is applicable to both animal and human brain tissue studies in neuropsychiatric research.

Application Note 24 describes stable isotope labelling in mammals (SILAM) using ¹⁵N-enriched spirulina to metabolically label rats and mice for quantitative proteomics. The note outlines diet preparation, labelling strategies (including generational labelling for rats and extended feeding protocols for mice), and experimental design in which ¹⁵N-labelled tissues serve as internal standards mixed 1:1 with unlabelled samples prior to processing. Quantitative analysis is performed using the Census algorithm to calculate ¹⁴N/¹⁵N peptide ratios, normalize data, and filter outliers. Applications highlighted include large-scale quantification of developmental proteome and phosphoproteome changes in brain tissue, demonstrating SILAM as a powerful in vivo approach for accurate, system-wide protein and post-translational modification analysis.

Application Note 23 describes IDAWG (Isotopic Detection of Aminosugars with Glutamine), a metabolic labelling strategy for glycomics that introduces ¹⁵N into glycans via supplementation of cell culture media with L-glutamine (amide-¹⁵N, 98%). Because the amide nitrogen of glutamine is the sole nitrogen donor in the hexosamine biosynthetic pathway, all GlcNAc-, GalNAc-, and sialic acid–containing glycans incorporate ¹⁵N, producing predictable mass shifts (+1 Da per aminosugar) detectable by high-resolution MS. Demonstrated in murine embryonic stem cells, the method achieved ~96–98% labelling efficiency and enables accurate comparative glycomic analysis and studies of glycan turnover without the limitations of radiotracer approaches.

Application Note 40 describes FLEXIQuant (Full-Length Expressed Stable Isotope-Labelled Proteins for Quantification), a strategy that uses in vitro–expressed ¹⁵N/¹³C-labelled full-length recombinant proteins as internal standards for absolute protein quantification and assessment of post-translational modifications. Heavy standards, produced using wheat germ expression systems with labelled lysine and arginine, are mixed with endogenous proteins prior to digestion and LC-MS analysis, where light/heavy peptide ratios reveal modification extent and dynamics. A built-in FLEX peptide enables absolute quantification via inverse isotope dilution MS. The method is demonstrated by tracking phosphorylation dynamics of CDC27 during mitotic arrest and is adaptable to kinase assays (FLEXIQinase) for mechanistic studies.

Application Note 50 describes the translation and implementation of the PeptiQuant™ Plus Human Plasma BAK-270 kit for large-scale, multiplexed protein quantification by LC-MRM/MS on a Shimadzu LC-MS platform. The kit enables targeted measurement of 270 plasma proteins using stable isotope–labelled internal standard peptides, with method optimization confirming robust detection across a wide dynamic range (mg/mL to sub-ng/mL). In validation studies, 213 interference-free peptides were reproducibly quantified in human plasma and serum, demonstrating suitability for complex clinical samples. The adapted method is being applied in clinical research at Montpellier Hospital, including a COVID-19 trial evaluating biomarkers of dexamethasone response, highlighting its utility for high-throughput biomarker discovery and clinical proteomics.

Application Note 54 describes the targeted MRM screening of U-¹³C crude lipid yeast extracts (L-ISO1) to support robust lipidomics workflows. Using rapid (8–12 min) RPLC- and HILIC-MRM/MS methods on a Xevo TQ-XS platform, the study verified a QC panel of 87 fatty acids and lipids across multiple classes—including glycerolipids, phospholipids, sphingolipids, and cardiolipins—and demonstrated additional detectable species beyond the core panel. Representative chromatograms (Figures 1–4) show strong signal intensity and resolution for DGs, PIs, PCs, and PEs, confirming reproducibility and broad coverage. The extract’s ¹³C enrichment ensures coelution with unlabelled counterparts, improving identification confidence and quantitative reliability, and supports applications in system suitability testing, credentialing, relative quantification, and method development for MS-based lipidomics.

Application Note 34 describes the use of D-glucose (U-¹³C₆, 99%) and L-glutamine (¹³C₅, 99%) to trace metabolic flux in cancer cells and mouse tumours. Using targeted LC-SRM/MS with polarity switching on a 5500 QTRAP platform, over 150 labelled metabolites were monitored to track ¹³C incorporation through glycolysis, the TCA cycle, the pentose phosphate pathway, and amino acid and nucleotide metabolism. In vivo labelling via intraperitoneal or jugular delivery revealed pathway-specific carbon utilization, with data showing that glutamine predominantly fuels the TCA cycle in pancreatic cancer. This isotope-tracing approach enables identification of altered metabolic pathways in tumours and supports the development of targeted metabolic therapies.

Application Note 31 describes the use of stable isotope-labelled fatty acids—such as ¹³C₁₈-oleic acid and perdeuterated palmitic, stearic, and oleic acids—to trace lipid synthesis and oxidation in vivo in mice. Using UPLC coupled to triple quadrupole or QTOF mass spectrometry, the study tracks incorporation of labelled oleate into triglycerides and cholesteryl esters and measures fatty acid oxidation via deuterium enrichment of plasma water. In a model of microsomal triglyceride transfer protein (MTP) inhibition, tracer-based analysis distinguished newly synthesized lipids from pre-existing pools, revealing acute effects on lipid assembly that were not evident from total lipid measurements alone. The approach improves sensitivity, expands the dynamic measurement window, reduces required animal numbers, and enables simultaneous assessment of lipid synthesis and oxidation without perturbing endogenous metabolism.

Application Note 30 describes a stable isotope tracer methodology for quantifying whole-body nitric oxide (NO) production and de novo arginine synthesis by measuring arginine–citrulline interconversion. Using L-arginine·HCl (guanido-¹⁵N₂, 98%) to trace NO formation (via production of ureido-¹⁵N citrulline) and labelled citrulline tracers to determine rates of appearance and conversion, the approach enables sensitive LC-MS/MS detection of low enrichment levels. The method accounts for tracer recycling and uses derivatisation to enhance analytical sensitivity. In mouse endotoxemia models, this strategy revealed time-dependent increases in NO production after LPS challenge and demonstrated a linear relationship between arginine supplementation and NO output, validating its utility for studying arginine metabolism and immune responses in vivo.

Application Note 29 describes a stable isotope labelling strategy to measure protein turnover in fish using L-leucine (isopropyl-D₇, 98%) incorporated into the diet. Common carp were fed a diet in which 50% of natural leucine was replaced with D₇-leucine, and incorporation into skeletal muscle proteins—specifically β-parvalbumin isoforms—was monitored by LC-MS of signature tryptic peptides. Using mass isotopomer distribution analysis, first-order synthesis rate constants were calculated, revealing that different parvalbumin isoforms exhibit markedly different turnover rates under steady-state conditions. The approach enables quantitative assessment of proteome dynamics in fish and can be extended to multiple proteins and species.

Application Note 52 describes a multiplexed LC-MRM/MS method for quantitative analysis of 16 bile acids (BAs) in mouse liver and ileum using CIL’s stable isotope-labelled bile acid mixes as internal standards. The method achieves baseline separation of isobaric BAs with a 6-point calibration curve spanning three orders of magnitude and demonstrates strong system suitability and negligible carryover. Applied to a murine myosin Vb (myoVb) knockout model of microvillus inclusion disease (MVID), the study revealed significantly decreased levels of several primary and conjugated BAs (including CA, DCA, β-MCA, GCA, TUDCA, TCDCA, and TCA) in liver, alongside increased CA and β-MCA in ileum. These findings suggest that loss of myoVb disrupts bile acid generation, transport, and recycling, providing mechanistic insight into cholestasis etiology and highlighting the utility of robust isotope-based BA quantification in liver disease research.

Application Note 51 demonstrates the use of QReSS™ stable isotope-labelled metabolite mixes as quality control (QC) and quantitation tools in LC-MS–based metabolomic analysis of foetal bovine serum (FBS). Using a chemically diverse panel of 18 labelled metabolites, the study assessed instrument performance in HILIC-MS workflows, showing excellent retention time stability (<1% CV), sub-ppm mass accuracy, and consistent signal response across >100 injections. Forward calibration curves enabled absolute quantitation of endogenous metabolites in FBS, revealing strong linearity (R² > 0.999) and reproducible measurements (<5% CV in pooled QCs). Comparative analysis showed that dialyzed FBS contained substantially lower metabolite levels than heat-inactivated or unprocessed samples, and untargeted profiling further highlighted pathway-level differences (e.g., choline synthesis) linked to processing method and geographic origin. Overall, the work underscores the importance of robust QC standards for reliable metabolite profiling and quantification in cell culture applications.

Application Note 49 describes the development and application of the QReSS™ (Quantification, Retention, and System Suitability) kit, which contains two reproducibly prepared mixes comprising 18 stable isotope-labelled metabolites designed to standardise LC-MS–based metabolomics workflows. The metabolites (detailed in Tables 1–3) span diverse classes and retention times, enabling broad chromatographic and mass spectrometric coverage. The note demonstrates their utility for system suitability testing—tracking retention time, peak shape, response, and mass accuracy over time—as well as for metabolite identification and relative/absolute quantification in plasma and urine using both targeted MRM and high-resolution SWATH-MS approaches. Overall, QReSS provides a robust QC and internal standard solution to improve reproducibility, performance monitoring, and quantitative reliability in metabolomics analyses.

Application Note 47 describes the development and validation of a targeted ion chromatography–tandem mass spectrometry (IC-MS/MS) method for quantifying polar, low molecular weight organic acids (OAs) in mouse quadricep muscle using stable isotope-labelled internal standards. Following derivatisation and separation on an IonPac AS11-HC column, 28 OAs were quantified with strong linearity (R² ≥ 0.99), good precision (~7% CV), average accuracies near 100%, and LOQs ranging from 0.25–50 µM. Applied to sedentary and exercise-fatigued mice, the method revealed statistically significant differences in hippurate, malate, fumarate, and α-ketoglutarate levels, demonstrating that IC-MS with isotopically labelled standards enables precise and accurate profiling of metabolically relevant organic acids in complex tissues.

Application Note 44 (Thermo Application Note 622) describes a high-throughput, pathway-targeted metabolomics workflow using ion chromatography coupled to high-resolution, accurate-mass Orbitrap MS (IC–Q Exactive HF) for quantitative analysis of TCA cycle intermediates in oral/head and neck cancer cells. Six stable isotope–labelled internal standards (¹³C- or D-labelled pyruvate, succinate, malate, fumarate, α-ketoglutarate, and citrate) were spiked into cell lysates to generate matrix-matched calibration curves spanning five orders of magnitude (0.1–10,000 pg/µL; R² ≥ 0.99). The IC method achieved strong resolution of polar metabolites—including challenging sugar mono- and diphosphates—with excellent retention time stability (±0.03 min over 150 injections). Targeted quantitation revealed that highly invasive cancer cells exhibit elevated TCA metabolite levels and that cancer stem-like cells show upregulation of first-half TCA intermediates (e.g., pyruvate, citrate, α-ketoglutarate) and downregulation of second-half intermediates (e.g., succinate, fumarate, malate), confirming prior untargeted findings. The study highlights the robustness of isotope-assisted IC-HRAM MS for large-scale, reproducible, and biologically informative metabolomics.

Application Note 21 describes the development of hyperpolarised metabolic contrast agents using PASADENA (Parahydrogen And Synthesis Allow Dramatically Enhanced Nuclear Alignment) to overcome the low sensitivity of conventional ¹³C and ¹⁵N NMR/MRI. The method uses parahydrogen addition to unsaturated ¹³C-labelled precursors (e.g., 1-¹³C-fumaric acid-D₂) followed by rapid spin-order transfer in a low magnetic field to generate highly polarised products such as 1-¹³C-succinic acid-D₂, achieving signal enhancements of ~100,000-fold. Deuteration extends ¹³C T₁ relaxation times (e.g., from ~6 to 27 seconds, and up to 56 seconds in D₂O), enabling in vivo metabolic imaging. The note highlights applications in cancer research, particularly for probing TCA cycle metabolism and succinate dehydrogenase (SDH) dysfunction, and discusses practical considerations including catalyst removal, pH optimisation, and automation for clinical translation.

Application Note 36 describes a cell-free protein synthesis strategy for producing uniformly ²H/¹⁵N/¹³C-labelled (perdeuterated) proteins in H₂O using labelled amino acids, eliminating the need for problematic ²H–¹H back-exchange required in conventional in vivo expression. By using E. coli S30 extracts and suppressing residual PLP-dependent enzyme activity with NaBH₄ treatment, unwanted ²H–¹H scrambling at α-positions is minimised. Comparison of [¹⁵N,¹H]-TROSY spectra (Figure 1) shows improved peak number and narrower line widths for proteins produced by cell-free synthesis versus in vivo expression in D₂O, particularly for unstable or non-refoldable proteins such as IMP-1 metallo-β-lactamase. The method yields milligram quantities of protein at competitive cost and is especially advantageous for large proteins (>40 kDa) used in advanced NMR studies.

Application Note 22 describes a novel isotopic labelling strategy using [2,3-¹³C]-labelled aromatic residues (Tyr and Phe) combined with uniformly ¹³C/¹⁵N-labelled Ala and Gly to improve signal intensity and facilitate resonance assignment of large membrane proteins by solid-state MAS-NMR. Applied to the 281-residue membrane protein OmpG, this “GAFY” labelling scheme reduces J-couplings and prevents magnetisation loss into aromatic sidechains, significantly enhancing Cα/Cβ crosspeak intensities in broadband PDSD spectra (as shown in Figure 2). The approach increases inter-residue correlations while maintaining small, isolated spin systems, enabling efficient sequence-specific assignments (illustrated for the GGF motif in Figure 3). Compared to glycerol-based labelling or band-selective experiments, the method improves spectral quality without excessive RF heating and supports more comprehensive structure determination of challenging membrane proteins.

Application Note 20 describes the optimisation of site-specific isotopic labelling (¹⁵N/¹³C-glycine, ¹⁵N/¹³C-phenylalanine, and ¹⁵N-tryptophan) of a recombinant human serine/threonine kinase expressed in baculovirus-infected SF9 insect cells using CIL’s BioExpress® 2000 media. A Box-Behnken Design of Experiment (DOE) approach systematically evaluated multiplicity of infection (MOI), harvest time (HPI), and cell density in small-scale (2 mL) screens to model and maximize protein yield. Statistical analysis (R² ≈ 80%, significant regression, no lack-of-fit) predicted optimal conditions (MOI 5, 48 h post-infection, 1.5 × 10⁶ cells/mL), which were validated at 0.5 L scale and then applied to 1 L Wave bioreactor production. The optimized process achieved ~1.3 mg/L labelled protein, representing ~20–40% improvement over screened conditions, demonstrating that DOE-guided optimisation reduces labelled media consumption while maximising yield for NMR sample preparation.

Application Note 19 describes the development of a ²H₂O-based E. coli cell-free (D-S30) extract system for efficient in vitro synthesis of highly perdeuterated proteins for NMR studies. Using [U-²H, 98%; U-¹⁵N, 98%] amino acids, the method achieved ~95% deuteration of nonlabile hydrogens, with translation efficiencies of ~65–85% relative to H₂O-based extracts (improved by ribosome supplementation). NMR analyses of FKBP (14 kDa) and GroEL (800 kDa, 14-mer) confirmed proper folding and assembly, with 2D [¹⁵N,¹H]-CRIPT-TROSY spectra of in vitro–synthesised GroEL closely matching in vivo samples. Amino acid–dependent back-protonation at α/β positions was observed (Table 1), but overall deuteration levels remained high. The approach enables economically viable production of perdeuterated proteins and supports advanced, selective isotope-labelling strategies for structural biology and drug discovery applications.

Application Note 15 provides practical guidance for maximizing yields of uniformly ¹³C/¹⁵N-labelled proteins in E. coli, illustrated through optimisation of DsbA C33S expression. The “top ten tips” cover controlling promoter leakiness (e.g., increasing lac repressor levels), balancing transcription/translation rates by adjusting strain (BL21(DE3), C41/C43(DE3)) and temperature, improving media composition (phosphate-buffered Studier Medium P, trace metals, optimised MgSO₄ and NH₄Cl), increasing glucose concentration (4 g/L significantly boosting yield), enhancing aeration, optimising induction timing and harvest time (e.g., extended 25°C expression), and using high-density resuspension strategies for labelling efficiency. The note also emphasises recovering all fractions during periplasmic extraction to avoid protein loss. Applying these strategies enabled production of ~100 mg/L labelled protein suitable for high-resolution solid-state NMR analysis, demonstrating broadly applicable methods for improving recombinant protein yield.

Application Note 14 describes efficient uniform ¹³C/¹⁵N isotope labelling of recombinant proteins expressed in baculovirus-infected Sf9 insect cells using BioExpress® 2000 insect cell media. Using the catalytic domain of Abl kinase as a model, the study demonstrates high expression levels (up to 115mg/L total protein; 50–80mg/L purified) and high isotopic incorporation (~90–91%) in both ¹⁵N- and ¹³C/¹⁵N-labelled media. High-quality ¹H–¹⁵N HSQC spectra confirmed proper folding and labelling efficiency. The optimised protocol involves growing cells initially in unlabelled medium, then transferring to labelled BioExpress 2000 prior to infection, significantly improving yield compared to continuous growth in labelling medium. The method enables production of >10 mg of correctly folded labelled protein and extends isotope-labelling capabilities to more complex eukaryotic proteins not readily expressed in E. coli, supporting advanced NMR studies.

Application Note 26 describes a cost-effective strategy for uniform ¹³C/¹⁵N isotope labelling of eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris, extending established labelling protocols from soluble proteins to seven-transmembrane targets. Using ¹³C₆-glucose during preinduction and ¹³C-methanol during induction (with ¹⁵NH₄₄Cl as nitrogen source), the authors achieved >90% isotopic incorporation and yields >5 mg/L of purified, functional Leptosphaeria rhodopsin (~31 kDa). Spectroscopic analyses—including FTIR and high-field (600–800 MHz) solid-state MAS NMR—demonstrated structural homogeneity, correct folding, minimal glycosylation, and well-resolved 2D ¹³C– ¹³C and NCOCX spectra suitable for resonance assignment and structural studies. The work establishes Pichia pastoris as a practical, scalable system for producing uniformly labelled eukaryotic membrane proteins for high-resolution NMR applications.

Application Note 48 describes stereospecific ¹³CH₃ labelling of Leu and Val methyl groups as a powerful strategy for solution NMR studies of very high–molecular-weight protein complexes. In highly deuterated backgrounds, selective labelling of only one prochiral methyl group (proR or proS) using specifically synthesised acetolactate precursors reduces spectral crowding and doubles sensitivity compared to racemic labelling, which splits signal intensity between two methyl positions. The approach is particularly valuable for methyl-TROSY experiments on complexes exceeding 100 kDa, including megadalton assemblies. Demonstrated on large systems such as the proteasome and the 320 kDa hexameric p97 fragment, stereospecific labelling enabled high-resolution ¹³C– ¹H HMQC spectra and revealed disease-related conformational dynamics not observable by X-ray crystallography alone.

Application Note 39 describes the production of U-[ ²²H], Thr-γ2[ ¹³CH₃]-labelled proteins for methyl-TROSY NMR studies of high–molecular-weight complexes. Extending methyl-labelling strategies beyond Ile, Leu, and Val, the method introduces a ¹³CH₃ label specifically at the threonine γ2 position in an otherwise highly deuterated background, improving spectral quality for large systems. The recommended protocol combines U-[²H], Thr-γ2[ ¹³CH₃] (50 mg/L), ¹³CH₃-labelled α-ketobutyrate (50 mg/L), and d₅-glycine (100 mg/L) to achieve optimal Thr and Ile (δ1) methyl labelling. Comparative spectra shown in Figure 1 (page 2) demonstrate markedly improved signal clarity in a 670 kDa proteasome sample prepared with the deuterated Thr precursor versus U-[ ¹³C, ¹H]-Thr, with the catalytic Thr1 methyl resonance only visible in the deuterated sample. This labelling strategy enables detailed structural and mechanistic studies of large molecular machines, including proteasomes and HslV proteases.

Application Note 35 discusses the role of biomonitoring in human exposure assessment and the analytical challenges associated with achieving attogram-level sensitivity for persistent organic pollutants. The note contrasts external dose modelling with internal dose measurement (biomonitoring), highlighting findings from studies such as the US Air Force Ranch Hand investigation, where serum TCDD levels were poorly correlated with modelled exposure indices (Figure 1, page 2). It reviews the CDC’s NHANES biomonitoring program and emphasizes the importance of isotope-dilution mass spectrometry using high-purity ¹³C-labelled standards for accurate quantification (Figures 4a and 4b illustrate improved accuracy when labelled internal standards are used). Advances such as cryogenic zone compression coupled with high-resolution MS now allow detection of 2,3,7,8-TCDD at ~313 attograms (Figure 5, page 4), corresponding to ~5.9 × 10⁵ molecules (Table 2). At these sensitivity levels, even trace impurities in analytical standards (e.g., 0.00001% in 1 ng equals 100 attograms) become detectable, underscoring the need for extremely high-purity unlabelled and isotopically labelled reference materials in modern biomonitoring studies.