Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM).

Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).

Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics.

The use of stable isotope-labeled standards (SIS) is an analytically valid means of quantifying proteins in biological samples. The nature of the labeled standards and their point of insertion in a bottom-up proteomic workflow can vary, with quantification methods utilizing curves in analytically sound practices. A promising quantification strategy for low sample amounts is external standard addition (ExSTA). In ExSTA, multipoint calibration curves are generated in buffer using serially diluted natural (NAT) peptides and a fixed concentration of SIS peptides. Equal concentrations of SIS peptides are spiked into experimental sample digests, with all digests (control and experimental) subjected to solid-phase extraction prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Endogenous peptide concentrations are then determined using the regression equation of the standard curves. Given the benefits of ExSTA in large-scale analysis, a detailed protocol is provided herein for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in undepleted and non-enriched human plasma samples. The procedural details and recommendations for successfully executing all phases of this quantification approach are described. As the proteins have been putatively correlated with various noncommunicable diseases, quantifying these by ExSTA in large-scale studies should help rapidly and precisely assess their true biomarker efficacy.

Highly accurate and precise quantification strategy using stable isotope dimethyl labeling coupled with GeLC-MS/MS.

Shotgun proteomics is a powerful method for comprehensively identifying and quantifying tryptic peptides, but it is difficult to analyze proteolytic events. One-dimensional gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) enables the separation of proteolytic fragments using SDS-PAGE followed by identification using LC-MS/MS. GeLC-MS/MS is thus an excellent method for identifying fragmentation. However, the lower reproducibility of gel extraction and nano flow LC-MS/MS can produce inaccurate results in comparative analyses of protein quantification among samples. In this study, a novel GeLC-MS/MS method coupled with stable isotope dimethyl labeling was developed. In the method, a mixture of light- and heavy-labeled samples is loaded onto an SDS-PAGE gel, and proteins with different isotopes in one extracted band are quantitatively analyzed by one-shot injection. This procedure enables accurate determination of the abundance ratio of peptides between two samples, even in cases of low peptide abundance, and it is not affected by the reproducibility of the gel extraction or LC-MS procedures. Therefore, our new GeLC-MS/MS method coupled with stable isotope dimethyl labeling provides high accuracy and comprehensive peptide comparisons, enabling the detection of proteolysis events caused by disease or physiological processes.

New Approaches for Absolute Quantification of Stable-Isotope-Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag.

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re-quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.

[The radiolabeled blood cell: Finding a legal qualification]. / La cellule sanguine marquée par un radionucléide : recherche d'une qualification juridique.

Radiolabelling of blood cells is a technique commonly used as a diagnostic tool in nuclear medicine; it has never been legally defined. This lack of legal status is a factor of uncertainty for both patients and health care professionals. The aim of this work was to identify what could be the legal nature of the radiolabelled blood cells by comparing their constitutive elements to the various existing legal categories applicable, according to the law applicable to living organisms as well as the regulations for other health products. The study concludes that, as it stands, the radiolabelled blood cells undoubtedly belong to the category of a drug by function for diagnostic purpose. More precisely, it is compared to a radiopharmaceutical medicinal product resulting from a well characterised manufacturing process. In order to increase visibility and thus the actors' awareness of the constraints arising from this specific status, it is proposed to create a specific legal regime for the radiolabelled blood cells by including in Article L. 5121-1 of the French Public Health Code a new category of health product which could be called: radiopharmaceutical preparation of cellular blood component for diagnostic purposes. The consequence of this proposal will mechanically place the radiolabelled blood cell preparation under the exclusive competences of radiopharmacists practising in an hospital pharmacy. Another important consequence will be that the radiolabelling process of blood cells will have to fulfil the rules of the French good hospital pharmacy practices and good preparation practices, for the benefit of patient protection and safety.

Quality control of imbalanced mass spectra from isotopic labeling experiments.

BACKGROUND: Mass spectra are usually acquired from the Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for isotope labeled proteomics experiments. In such experiments, the mass profiles of labeled (heavy) and unlabeled (light) peptide pairs are represented by isotope clusters (2D or 3D) that provide valuable information about the studied biological samples in different conditions. The core task of quality control in quantitative LC-MS experiment is to filter out low-quality peptides with questionable profiles. The commonly used methods for this problem are the classification approaches. However, the data imbalance problems in previous control methods are often ignored or mishandled. In this study, we introduced a quality control framework based on the extreme gradient boosting machine (XGBoost), and carefully addressed the imbalanced data problem in this framework. RESULTS: In the XGBoost based framework, we suggest the application of the Synthetic minority over-sampling technique (SMOTE) to re-balance data and use the balanced data to train the boosted trees as the classifier. Then the classifier is applied to other data for the peptide quality assessment. Experimental results show that our proposed framework increases the reliability of peptide heavy-light ratio estimation significantly. CONCLUSIONS: Our results indicate that this framework is a powerful method for the peptide quality assessment. For the feature extraction part, the extracted ion chromatogram (XIC) based features contribute to the peptide quality assessment. To solve the imbalanced data problem, SMOTE brings a much better classification performance. Finally, the XGBoost is capable for the peptide quality control. Overall, our proposed framework provides reliable results for the further proteomics studies.

Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay.

Stable isotope-labeled standard (SIS) peptides are used as internal standards in targeted proteomics to provide robust protein quantification, which is required in clinical settings. However, SIS peptides are typically added post trypsin digestion and, as the digestion efficiency can vary significantly between peptides within a protein, the accuracy and precision of the assay may be compromised. These drawbacks can be remedied by a new class of internal standards introduced by the Human Protein Atlas project, which are based on SIS recombinant protein fragments called SIS PrESTs. SIS PrESTs are added initially to the sample and SIS peptides are released on trypsin digestion. The SIS PrEST technology is promising for absolute quantification of protein biomarkers but has not previously been evaluated in a clinical setting. An automated and scalable solid phase extraction workflow for desalting and enrichment of plasma digests was established enabling simultaneous preparation of up to 96 samples. Robust high-precision quantification of 13 apolipoproteins was achieved using a novel multiplex SIS PrEST-based LC-SRM/MS Tier 2 assay in non-depleted human plasma. The assay exhibited inter-day coefficients of variation between 1.5% and 14.5% (median = 3.5%) and was subsequently used to investigate the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on these 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECT I (NCT02354976). No significant changes were observed in the OM3-CA arm, whereas treatment with fenofibrate significantly increased apoAII and reduced apoB, apoCI, apoE and apoCIV levels. The reduction in apoCIV following fenofibrate treatment is a novel finding. The study demonstrates that SIS PrESTs can facilitate the generation of robust multiplexed biomarker Tier 2 assays for absolute quantification of proteins in clinical studies.

Rapidly Assessing the Quality of Targeted Proteomics Experiments through Monitoring Stable-Isotope Labeled Standards.

Targeted proteomics experiments based on selected reaction monitoring (SRM) have gained wide adoption in the use of clinical biomarkers, cellular modeling, and numerous other biological experiments due to their highly accurate and reproducible quantification. The quantitative accuracy in targeted proteomics experiments is reliant on the stable-isotope, heavy-labeled peptide standards that are spiked into a sample and used as a reference when calculating the abundance of endogenous peptides. Therefore, the quality of measurement for these standards is a critical factor in determining whether data acquisition was successful. With improved mass spectrometry (MS) instrumentation that enables the monitoring of hundreds of peptides in hundreds to thousands of samples, quality assessment is increasingly important and cannot be performed manually. We present Q4SRM, a software tool that rapidly checks the signal from all heavy-labeled peptides and flags those that fail quality-control metrics. Using four metrics, the tool detects problems with both individual SRM transitions and the collective group of transitions that monitor a single peptide. The program's speed and simplicity enable its use at the point of data acquisition and can be ideally run immediately upon the completion of a liquid chromatography-SRM-MS analysis.

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.

Metabolite Spectral Accuracy on Orbitraps.

Orbitraps are high-resolution ion-trap mass spectrometers that are widely used in metabolomics. While the mass accuracy and resolving power of orbitraps have been extensively documented, their spectral accuracy, i.e., accuracy in measuring the abundances of isotopic peaks, remains less studied. In analyzing spectra of unlabeled metabolites, we discovered a systematic under representation of heavier natural isotopic species, especially for high molecular weight metabolites (∼20% under-measurement of [M + 1]/[M + 0] ratio at m/z 600). We hypothesize that these discrepancies arise for metabolites far from the lower limit of the mass scan range, due to the weaker containment in the C-trap that results in suboptimal trajectories inside the Orbitrap analyzer. Consistent with this, spectral fidelity was restored by dividing the mass scan range (initially 75 m/z to 1000 m/z) into two scan events, one for lower molecular weight and the other for higher molecular weight metabolites. Having thus obtained accurate mass spectra at high resolution, we found that natural isotope correction for high-resolution labeling data requires more sophisticated algorithms than typically employed: the correction algorithm must take into account whether isotopologues with the same nominal mass are resolved. We present an algorithm and associated open-source code, named AccuCor, for this purpose. Together, these improvements in instrument parameters and natural isotope correction enable more accurate measurement of metabolite labeling and thus metabolic flux.

Quality control on radiochemical purity in Technetium-99m radiopharmaceuticals labelling: three years of experience on 2280 procedures.

OBJECTIVE: the purpose of this study was to offer an example of evaluations of the ISO9001 certified internal quality assurance (QA) system of 99mTc-radiopharmaceutical preparations and quality control in vivo use, using industrial kits and generators in order to identify possible sources of errors in the procedures labeling and quality control procedures. METHODS: The study was performed at a single institution over a period of three years (July 1st, 2011 - July 1st, 2014), and included a total of 2280 radiopharmaceutical preparations prepared by four different technologists. All the radiopharmaceutical preparations and quality controls were performed according to each SPC provided by the manufacturer. The radiopharmaceutical preparations were the following (trade names are reported in brackets): 99mTc-albumin colloid [Nanocoll] (n=349), 99mTc-oxidronate [Technescan®hdp] (n=701), 99mTc-exametazime [Ceretec] (n=169), 99mTc-sestamibi [Cardiolite] (n=92), 99mTc-albumin aggregated [Technescan®lyomaa] (n=140), 99mTc-tetrofosmin [Myoview]) (n=567), 99mTc-diethylene triamine pentacetic acid [Technescan®dtpa] (n=254), and 99mTc-dimercapto succinic acid [Renocis®] (n=8). Data were analyzed to determine the number and type of radiopharmaceutical labelling failure and to derive the sources of these failures to define corrective actions and optimize the quality assurance program. RESULTS: A total of 2280 procedures were performed and recorded. Following the quality control procedure six out of the 2280 preparations (0.26%) were non-conforming for clinical use with the RCP limits indicated in the SPC. Five of these were due to gross technical errors in measurements and manual procedures and were immediately repeated, returning within the limits of acceptability. The sixth failure was due to short incubation time, though compliant with the manufacturer's instructions. CONCLUSIONS: We concluded that the quality of the final product depends on a controlled production system based on the implementation of specific standard operating procedures (ISO9001, SOP) for each radiopharmaceutical production, according to strict adherence to the SPC of each radiopharmaceutical. Based on these conclusions, in our opinion every quality control suggesting a possible error in the synthesis procedure recommended in the SPC should be immediately reported to the manufacturer, for a revision of the SPC, as well as to the regulatory agencies for an alert. This strategy may in fact allow the continuous improvement of the labelling procedures and therefore the optimization of the quality control procedures frequency to ensure both patients safety and a more rational management of resources for economic sustainability.

Optimization of Labeling PSMAHBED with Ethanol-Postprocessed 68Ga and Its Quality Control Systems.

Radiolabeling of the prostate-specific membrane antigen (PSMA) inhibitor Glu-NH-CO-NH-Lys(Ahx) using the 68Ga chelator HBED-CC (PSMAHBED) allows imaging of prostate cancer lesions because of high expression of PSMA in prostate carcinoma cells and in bone metastases and lymph nodes related to the disease. The aim of this work was to optimize labeling of 68Ga-PSMAHBED using the efficient cation-exchange postprocessing of 68Ga as well as the development of a thin-layer chromatography (TLC)-based quality control system. Methods: Labeling was optimized for online ethanol-postprocessed 68Ga eluate investigating various parameters, such as buffer molarity (0.1-1 M), temperature (25°C-90°C), tracer amount (0.11-0.74 nmol), and labeling time. In addition, purification of the crude product was tested. For radio-TLC quality control, various mobile phases were analyzed using silica gel 60 plates and the results were validated using high-performance liquid chromatography. The most superior mobile phases were also applied on instant thin-layer chromatography (ITLC) silica gel plates. Results: Using optimized conditions, labeling yields of more than 95% were obtained within 10 min when ethanol-based postprocessing was applied using PSMAHBED amounts as low as 0.1 nmol. A higher precursor concentration (0.7 nmol) further increased labeling and quantitative yields to more than 98% within 5 min. In clinical routine, patient batches (>200 applications) with radiochemical purity greater than 98% and specific activities of 326 ± 20 MBq/nmol are obtained reproducibly. When TLC quality control was performed on silica gel 60 plates, 4 mobile phases with suitable separation properties and complementary Rf values were identified. Two systems showed equivalent separation on ITLC silica gel plates, with ITLC analysis finished within 5 min, in contrast to 20 min for the TLC system. Labeling of PSMAHBED was optimized for cation-exchange postprocessing methods, ensuring almost quantitative labeling and high nuclide purity of final 68Ga-PSMAHBED, making subsequent purification steps unnecessary. Conclusion: The new radio-TLC method allows quality control in a short time using a fast, reliable, low-cost method with little equipment complexity. Using this approach, the synthesis is easily adopted by automated synthesis modules.

Stable Isotope Labeling for Improved Comparative Analysis of RNA Digests by Mass Spectrometry.

Even with the advent of high throughput methods to detect modified ribonucleic acids (RNAs), mass spectrometry remains a reliable method to detect, characterize, and place post-transcriptional modifications within an RNA sequence. Here we have developed a stable isotope labeling comparative analysis of RNA digests (SIL-CARD) approach, which improves upon the original 18O/16O labeling CARD method. Like the original, SIL-CARD allows sequence or modification information from a previously uncharacterized in vivo RNA sample to be obtained by direct comparison with a reference RNA, the sequence of which is known. This reference is in vitro transcribed using a 13C/15N isotopically enriched nucleoside triphosphate (NTP). The two RNAs are digested with an endonuclease, the specificity of which matches the labeled NTP used for transcription. As proof of concept, several transfer RNAs (tRNAs) were characterized by SIL-CARD, where labeled guanosine triphosphate was used for the reference in vitro transcription. RNase T1 digestion products from the in vitro transcript will be 15 Da higher in mass than the same digestion products from the in vivo tRNA that are unmodified, leading to a doublet in the mass spectrum. Singlets, rather than doublets, arise if a sequence variation or a post-transcriptional modification is present that results in a relative mass shift different from 15 Da. Moreover, the use of the in vitro synthesized tRNA transcript allows for quantitative measurement of RNA abundance. Overall, SIL-CARD simplifies data analysis and enhances quantitative RNA modification mapping by mass spectrometry. Graphical Abstract ᅟ.

68Ga 'vacuum elution approach' for direct radiolabelling of DOTA-conjugated and NODAGA-conjugated peptides.

BACKGROUND/OBJECTIVES: The Ge/Ga generator is of increasing interest for clinical PET. The arrival on the market of the pharmaceutical-grade generator, which provides an eluate with chemical and radiochemical purities in conformity with the European Pharmacopeia specifications, makes the direct labelling of vectors possible. The kit formulation strategies using single vial productions can improve the access of hospitals and imaging centres that are not equipped with costly automated synthesis modules to the Ga-radiopharmaceutical production. The manual radiosynthesis of Ga requires handling of a relatively high amount of radioactivity, resulting in a high radiation dose to the hand. Moreover, the elution of the Ga/Ge generator with 5 ml of HCl as recommended by the manufacturer leads to a low Ga concentration, which can decrease the efficiency of the labelling procedure. The aim of our approach is to circumvent these disadvantages and to offer an alternative to the hand elution and labelling for a routine production of Ga-radiopharmaceuticals. METHODS: A mixture of buffer and peptide was first transferred to an evacuated collection vial. Fixed volume of HCl was adapted to the inlet line of the generator. The elution was then performed by the action of vacuum and the labeling occurs at RT or 95°C. RESULTS AND CONCLUSION: The 'vacuum elution approach' developed in this work enables the elution of 95% of the available generator activity with 2.5 ml of eluent, the direct labelling of DOTA-conjugated and NODAGA-conjugated peptides with high radiochemical (>97% for all cases) and radionuclidic (100%) purities without exposure of the hand to radiation during the preparation steps.

Comprehensive Quality Control of the ITG 68Ge/68Ga Generator and Synthesis of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC for Clinical Imaging.

UNLABELLED: A good-manufacturing-practices (GMP) (68)Ge/(68)Ga generator that uses modified dodecyl-3,4,5-trihydroxybenzoate hydrophobically bound to a octadecyl silica resin (C-18) as an adsorbent has been developed that allows for dilute HCl (0.05N) to efficiently elute metal-impurity-free (68)Ga(3+) ready for peptide labeling. We characterized the performance of this generator system over a year in conjunction with the production of (68)Ga-labeled DOTATOC and Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (PSMA-HBED-CC) intended for clinical studies and established protocols for batch release. METHODS: A 2,040-MBq self-shielded (68)Ge/(68)Ga generator provided metal-free (68)GaCl3 ready for peptide labeling in the fluidic labeling module after elution with 4 mL of 0.05N HCl. The compact system was readily housed in a laminar flow cabinet allowing an ISO class-5 environment. (68)Ga labeling of peptides using GMP kits was performed in 15-20 min, and the total production time was 45-50 min. Batch release quality control specifications were established to meet investigational new drug submission and institutional review board approval standards. RESULTS: Over a period of 12 mo, (68)Ga elution yields from the generator averaged 80% (range, 72.0%-95.1%), and (68)Ge breakthrough was less than 0.006%, initially decreasing with time to 0.001% (expressed as percentage of (68)Ge activity present in the generator at the time of elution), a unique characteristic of this generator. The radiochemical purity of both (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC determined by high-performance liquid chromatography analysis was greater than 98%, with a minimum specific activity of 12.6 and 42 GBq/µmol, respectively. The radionuclidic ((68)Ge) impurity was 0.00001% or less (under the detection limit). Final sterile, pyrogen-free formulation was provided in physiologic saline with 5%-7% ethanol. CONCLUSION: The GMP-certified (68)Ge/(68)Ga generator system was studied for a year. The generator system is contained within the fluidic labeling module, and it is compact, self-shielded, and easy to operate using simple manual techniques. The system provides radiolabeled peptides with high (>98%) radiochemical purity and greater than 80% radiochemical yield. The (68)Ge levels in the final drug products were under the detection limits at all times. (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC investigational radiopharmaceuticals are currently being studied clinically under investigational new drug (IND) applications submitted to the U.S. Food and Drug Administration.

Recommended administered activities for (68)Ga-labelled peptides in paediatric nuclear medicine.

PURPOSE: The aim of this study was to establish a method for determining administered activities for (68)Ga-labelled peptides. Dose calculations were based on the weight-independent effective dose model proposed by the EANM paediatric dosage card for use in paediatric nuclear medicine. METHODS: Previously published time-integrated activity coefficients for (68)Ga-DOTATATE, (68)Ga-DOTATOC and (68)Ga-pentixafor were used to calculate age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card to determine the radiopharmaceutical class of  (68)Ga-labelled peptides ("multiples") and to calculate the baseline activities based on an upper limit for administered activity (185 MBq) in an adult. RESULTS: All calculated normalization factors suggest that the (68)Ga-labelled peptides are class "B" radiopharmaceuticals. The baseline activity for all compounds is 12.8 MBq. In analogy to (18)F-fluoride, we recommend a minimum activity of 14 MBq. CONCLUSION: For paediatric nuclear medicine applications involving (68)Ga-labelled peptides, we suggest determining administered activities based on the formalism proposed in this work. The corresponding effective doses from these procedures will remain age-independent.

Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation.

Protein quantification based on peptides using LC-MS/MS has emerged as a promising method to measure biomarkers, protein drugs, and endogenous proteins. However, the best practices for selection, optimization, and validation of the quantification peptides are not well established, and the influence of different matrices on protein digestion, peptide stability, and MS detection has not been systematically addressed. The aim of this study was to determine how biological matrices affect digestion, detection, and stability of peptides. The microsomal retinol dehydrogenase (RDH11) and cytosolic soluble aldehyde dehydrogenases (ALDH1As) involved in the synthesis of retinoic acid (RA) were chosen as model proteins. Considerable differences in the digestion efficiency, sensitivity, and matrix effects between peptides were observed regardless of the target protein's subcellular localization. The precision and accuracy of the quantification of RDH11 and ALDH1A were affected by the choice of calibration and internal standards. The final method using recombinant protein calibrators and stable isotope labeled (SIL) peptide internal standards was validated for human liver. The results demonstrate that different sample matrices have peptide, time, and matrix specific effects on protein digestion and absolute quantification.

Comparing the effect of isotopically labeled or structural analog internal standards on the performance of a LC-MS/MS method to determine ciclosporin A, everolimus, sirolimus and tacrolimus in whole blood.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used for analysis of immunosuppressive drugs. This study investigated whether replacing analog internal standards (ANISs) with isotopically labeled internal standards (ILISs) has an impact on the performance of a LC-MS/MS method for the quantification of tacrolimus (TAC), sirolimus (SIR), ciclosporin A (CsA) and everolimus (EVE) in whole blood. METHODS: Following hemolysis, protein precipitation, and extraction with either ANISs (ascomycin, desmethoxy-rapamycin, CsD), or ILISs (TAC-13C,D2; SIR-13C,D3; CsA-D12; EVE-D4), samples were centrifuged and injected into a LC-MS/MS device equipped with a C18 reversed phase column. The effect of the two ISs on the linearity, precision, accuracy, trueness, matrix effects, and carryover was investigated by using the same patient-, proficiency testing-, and quality control samples. Statistical analysis of agreement between results includes a standard random effects model and Passing-Bablok regression. RESULTS: Within-day imprecision was <10%, between-day <8%, and trueness 91%-110% for all the analytes with both ISs. No carryover or matrix effects were observed. The median accuracy was -2.1% for CsA, 9.1% for EVE, 12.2% for SIR, and -1.2% for TAC with the ILISs; and -2% for CsA, 9.8% for EVE, 11.4% for SIR, and 0.2% for TAC with the ANISs. Results of patient and proficiency testing samples were not statistically different. CONCLUSIONS: Although ILISs are generally considered superior to ANISs, they may not be always essential. When optimizing a LC-MS/MS method other factors must be also considered.

Methods for differential and quantitative analyses of brain neurosteroid levels by LC/MS/MS with ESI-enhancing and isotope-coded derivatization.

The analysis of changes in the brain neurosteroid (NS) levels due to various stimuli can contribute to the elucidation of their physiological roles, and the discovery and development of new antipsychotic agents targeting neurosteroidogenesis. We developed methods for the differential and quantitative analyses of the brain levels of allopregnanolene (AP) and its precursor, pregnenolone (PREG), using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using 2-hydrazino-1-methylpyridine (HMP) and its isotope-coded analogue, (2)H3-HMP (d-HMP). For the differential analysis, the brain sample of an untreated rat was derivatized with HMP, while the brain sample of a treated (stressed or drug-administered) rat was derivatized with d-HMP. The two derivatives were mixed and then subjected to LC/ESI-MS/MS. The stress- and drug (clozapine and fluoxetine)-evoked increases in the brain AP and PREG levels were accurately analyzed by the developed method. It was also possible to determine the absolute concentrations of the brain steroids when a deuterium-coded moiety was introduced to the standard steroids of known amounts by the derivatization and the resulting derivatives were used as internal standards. The HMP-derivatization enabled the highly sensitive detection and the use of d-HMP significantly improved the assay precision [the intra- (n=5) and inter-assay (n=5) relative standard deviations did not exceed 13.7%] and accuracy (analytical recovery ranged from 98.7 to 106.7%).