MALDI-MSI-LC-MS/MS Workflow for Single-Section Single Step Combined Proteomics and Quantitative Lipidomics.

We introduce a novel approach for comprehensive molecular profiling in biological samples. Our single-section methodology combines quantitative mass spectrometry imaging (Q-MSI) and a single step extraction protocol enabling lipidomic and proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on the same tissue area. The integration of spatially correlated lipidomic and proteomic data on a single tissue section allows for a comprehensive interpretation of the molecular landscape. Comparing Q-MSI and Q-LC-MS/MS quantification results sheds new light on the effect of MSI and related sample preparation. Performing MSI before Q-LC-MS on the same tissue section led to fewer protein identifications and a lower correlation between lipid quantification results. Also, the critical role and influence of internal standards in Q-MSI for accurate quantification is highlighted. Testing various slide types and the evaluation of different workflows for single-section spatial multiomics analysis emphasized the need for critical evaluation of Q-MSI data. These findings highlight the necessity for robust quantification methods comparable to current gold-standard LC-MS/MS techniques. The spatial information from MSI allowed region-specific insights within heterogeneous tissues, as demonstrated for glioblastoma multiforme. Additionally, our workflow demonstrated the efficiency of a single step extraction for lipidomic and proteomic analyses on the same tissue area, enabling the examination of significantly altered proteins and lipids within distinct regions of a single section. The integration of these insights into a lipid-protein interaction network expands the biological information attainable from a tissue section, highlighting the potential of this comprehensive approach for advancing spatial multiomics research.

Improved on-tissue detection of the anti-cancer agent doxorubicin by quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging.

Doxorubicin (dox) is an affordable, and highly effective chemotherapeutic agent used in cancer treatment, yet its application is known to cause cumulative cardiac and renal toxicity. In this study, we employed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to evaluate the distribution of dox in mouse heart and kidney after in vivo treatment. To this end, we performed absolute quantification using an isotopically labeled form (13C d3-dox) as an internal standard. Unfortunately, ion suppression often leads to loss of sensitivity in compound detection and can result in hampered drug quantification. To overcome this issue, we developed an on-tissue chemical derivatization (OTCD) method using Girard's reagent T (GirT). With the developed method, dox signal was increased by two orders of magnitude. This optimized sample preparation enabled a sensible gain in dox detection, making it possible to study its distribution and abundance (up to 0.11 pmol/mm2 in the heart and 0.33 pmol/mm2 in the kidney medulla). The optimized approach for on-tissue derivatization and subsequent quantification creates a powerful tool to better understand the relationship between dox exposure (at clinically relevant concentrations) and its biological detrimental effects in various tissues. Overall, this work is a showcase of the added value of MALDI-MSI for pharmaceutical studies to better understand heterogeneity in drug exposure between and within organs.

Toward Omics-Scale Quantitative Mass Spectrometry Imaging of Lipids in Brain Tissue Using a Multiclass Internal Standard Mixture.

Mass spectrometry imaging (MSI) has accelerated our understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously over the tissue surface with concentrations that reflect those of endogenous lipids. This enabled quantitative MSI (Q-MSI) of 13 lipid classes and subclasses representing almost 200 sum-composition lipid species using both MALDI (negative ion mode) and MALDI-2 (positive ion mode) and pixel-wise normalization of each lipid species in a manner analogous to that widely used in shotgun lipidomics. The Q-MSI approach covered 3 orders of magnitude in dynamic range (lipid concentrations reported in pmol/mm2) and revealed subtle changes in distribution compared to data without normalization. The robustness of the method was evaluated by repeating experiments in two laboratories using both timsTOF and Orbitrap mass spectrometers with an ∼4-fold difference in mass resolution power. There was a strong overall correlation in the Q-MSI results obtained by using the two approaches. Outliers were mostly rationalized by isobaric interferences or the higher sensitivity of one instrument for a particular lipid species. These data provide insight into how the mass resolving power can affect Q-MSI data. This approach opens up the possibility of performing large-scale Q-MSI studies across numerous lipid classes and subclasses and revealing how absolute lipid concentrations vary throughout and between biological tissues.

Extensive profiling of histidine-containing dipeptides reveals species- and tissue-specific distribution and metabolism in mice, rats, and humans.

AIM: Histidine-containing dipeptides (HCDs) are pleiotropic homeostatic molecules with potent antioxidative and carbonyl quenching properties linked to various inflammatory, metabolic, and neurological diseases, as well as exercise performance. However, the distribution and metabolism of HCDs across tissues and species are still unclear. METHODS: Using a sensitive UHPLC-MS/MS approach and an optimized quantification method, we performed a systematic and extensive profiling of HCDs in the mouse, rat, and human body (in n = 26, n = 25, and n = 19 tissues, respectively). RESULTS: Our data show that tissue HCD levels are uniquely produced by carnosine synthase (CARNS1), an enzyme that was preferentially expressed by fast-twitch skeletal muscle fibres and brain oligodendrocytes. Cardiac HCD levels are remarkably low compared to other excitable tissues. Carnosine is unstable in human plasma, but is preferentially transported within red blood cells in humans but not rodents. The low abundant carnosine analogue N-acetylcarnosine is the most stable plasma HCD, and is enriched in human skeletal muscles. Here, N-acetylcarnosine is continuously secreted into the circulation, which is further induced by acute exercise in a myokine-like fashion. CONCLUSION: Collectively, we provide a novel basis to unravel tissue-specific, paracrine, and endocrine roles of HCDs in human health and disease.

MALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined.

Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDI-IHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDI-IHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-αSM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.

Postmortem Analysis of Opioids and Metabolites in Skeletal Tissue.

Every year, thousands of suspicious deaths are accounted for by an overdose of opioids. Occasionally all traditional matrices are unavailable due to decomposition. Skeletal tissue may pose a valid alternative. However, reference data on postmortem concentrations in bone tissue and bone marrow (BM) is sparse. Therefore, a liquid chromatography--tandem mass spectrometry method was developed and fully validated for the analysis of four opioids and two metabolites (tramadol, O-desmethyltramadol, morphine, fentanyl, norfentanyl, codeine) in bone tissue and BM. Sample preparation was performed using solid phase extraction (BM), methanolic extraction (bone) and a protein precipitation (whole blood). All validation parameters were successfully fulfilled. This method was applied to analyze 22 forensic cases involving opioids. All six opioids were proven to be detectable and quantifiable in all specimens sampled. When tramadol blood concentrations were correlated with bone concentrations, a linear trend could be detected. The same was seen between tramadol blood and BM concentration. A similar linear trend was seen when correlating codeine blood concentration with bone and BM concentration. Although some variability was detected, the same linear trend was seen for morphine. For fentanyl and norfentanyl, the sample size was too small to draw conclusions, regarding correlation. As far as the authors know this is the first-time fentanyl and norfentanyl are quantified in skeletal tissue. In conclusion, due to the absence of reference data for drugs in skeletal tissue, these findings are a step forward toward a more thorough understanding of drug concentration found in postmortem skeletal tissue.

Sample preparation of bone tissue for MALDI-MSI for forensic and (pre)clinical applications.

In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.

Skeletal tissue, a viable option in forensic toxicology? A view into post mortem cases.

Blood analysis is the golden standard in the field of forensic toxicology. However, when extended decomposition of the remains has occurred, alternative matrices are required. Skeletal tissue may provide an appropriate sample of choice since it is very resistant to putrefaction. However, today, the absence of reference data of drug concentrations in skeletal tissue poses a problem to meaningfully and reliably conduct toxicological testing on human skeletal material. The present study investigates the viability of skeletal tissue as an alternative matrix to evaluate xenobiotic consumption in legal cases. Blood, bone tissue and bone marrow of different forensic cases were screened for 415 compounds of forensic interest. Afterwards, methadone, clomipramine, citalopram and their respectively metabolites positive samples were quantified using fully validated methods. Sample preparation was carried out by SPE (whole blood and bone marrow), methanol extraction (bone sections) or protein precipitation (whole blood). All samples were analyzed using liquid chromatography coupled to a triple quad mass spectrometer. Multiple drugs were successfully identified in all sampled matrices. In bone (marrow) not as many substances were detected as in blood but it poses a valid alternative when blood is not available. Especially bone marrow showed big potential with a concordance of 80.5% with blood. Clomipramine, citalopram and their metabolites were proven to be detectable and quantifiable in all specimens sampled. Bone marrow showed the highest concentrations followed by blood and bone tissue. When citalopram blood concentrations were correlated with the bone concentrations, a linear trend could be detected. The same was seen between blood and bone marrow for citalopram concentrations. Methadone was also proven to be detectable in all specimens sampled. However, its metabolites EMDP and EDPP were absent or below the LOD in some samples. Overall, methadone concentrations were higher in bone marrow than in bone. With exception of one case, blood concentrations were higher than bone concentrations. For methadone, a linear trend could be found between blood and bone concentration. Comparing methadone concentrations in blood and bone marrow an exponential trend could be seen. In conclusion, these findings show the potential forensic value of bone and bone marrow as an alternative matrix. Aside to that, a standard protocol for the sample collection and processing is proposed.

A Historical-Genetic Reconstruction of Human Extra-Pair Paternity.

Paternity testing using genetic markers has shown that extra-pair paternity (EPP) is common in many pair-bonded species [1, 2]. Evolutionary theory and empirical data show that extra-pair copulations can increase the fitness of males as well as females [3, 4]. This can carry a significant fitness cost for the social father, who then invests in rearing offspring that biologically are not his own [5]. In human populations, the incidence and correlates of extra-pair paternity remain highly contentious [2, 6, 7]. Here, we use a population-level genetic genealogy approach [6, 8] to reconstruct spatiotemporal patterns in human EPP rates. Using patrilineal genealogies from the Low Countries spanning a period of over 500 years and Y chromosome genotyping of living descendants, our analysis reveals that historical EPP rates, while low overall, were strongly impacted by socioeconomic and demographic factors. Specifically, we observe that estimated EPP rates among married couples varied by more than an order of magnitude, from 0.4% to 5.9%, and peaked among families with a low socioeconomic background living in densely populated cities of the late 19th century. Our results support theoretical predictions that social context can strongly affect the outcomes of sexual conflict in human populations by modulating the incentives and opportunities for engaging in extra-pair relationships [9-11]. These findings show how contemporary genetic data combined with in-depth genealogies open up a new window on the sexual behavior of our ancestors.

Distribution of clomipramine, citalopram, midazolam, and metabolites in skeletal tissue after chronic dosing in rats.

In recent years, the use of skeletal tissue as an alternative matrix in forensic toxicology has received new interest. In cases where extreme decomposition has taken place, analysis of skeletal tissue is often the only option left. In this article, a fully validated method is presented and the distribution of clomipramine, citalopram, midazolam, and metabolites after chronically administration is examined within skeletal tissue. Rats were chronically dosed with respectively clomipramine, citalopram, or midazolam. Extracts were quantitatively analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Clomipramine, citalopram, and metabolites, respectively desmethylclomipramine and desmethylcitalopram are shown to be detectable in all bone types sampled. Midazolam and its metabolite α-OH-midazolam could not be detected. The absence of midazolam in extracts gives an indication that drugs with pKa values under physiological pH are badly or not incorporated in bone tissue. Bone and post-mortem blood concentrations were compared. A range of different bone types was compared and showed that the concentration is strongly dependent on the bone type. In concordance with previous publications, the humerus shows the highest drug levels. Skeletal tissue concentrations found ranged from 1.1 to 587.8 ng/g. Comparison of the same bone type between the different rats showed high variances. However, the drugs-metabolite ratio proved to have lower variances (<20%). Moreover, the drugs-metabolite ratio in the sampled bones is in close concordance to the ratios seen in blood within a rat. From this, we can assume that the drugs-metabolite ratio in skeletal tissue may prove to be more useful than absolute found concentration.

Distribution of Methadone and Metabolites in Skeletal Tissue.

In forensic toxicology, when extended time has elapsed before discovery of the body, the most commonly analyzed specimens are often degraded or not available at all due to decomposition. In this case, skeletal tissue may be the only specimen left. Nevertheless, very limited research is found on the drug disposition in bone, making toxicological interpretation very difficult. Since methadone is linked to almost 50% of drug abuse fatalities in Belgium, an easy extraction and quantification method was developed and validated to investigate the distribution pattern of methadone and its metabolites in skeletal tissue after chronic dosing. In this study, Wistar rats were administered a subcutaneous daily methadone dose of 3 mg/kg for 139 days. After dissection, single whole bones or bone parts underwent a methanolic extraction. The final extract was analyzed using LC-ESI(+)-MS-MS for methadone, EDDP and EMDP. Methadone and its metabolites were proven to be detectable and quantifiable in skeletal tissue of chronically dosed rats using a fast and easy methanol extraction. Within bone, comparison showed that bone marrow yields the highest concentration. Trabecular bone also showed to be the best type of bone tissue for sampling. Between bone comparison, proved the humerus to be the best bone type for sampling. The concentrations found in tibiae and humeri appeared to be dose dependent for methadone with a variance of <9%. However, for other bones the variance in methadone concentration ranged from 24 to 32%. A possible explanation is seen in the lower vascularization of these bones. For the metabolites, no correlation was seen. This could be explained by the highly inter-individual metabolism of methadone. However, skeletal tissue concentration showed no correlation to blood for methadone nor its metabolites. Using the developed method, quantitative information about methadone after chronic administration was only found in the humeri and tibiae.

Determining Y-STR mutation rates in deep-routing genealogies: Identification of haplogroup differences.

Knowledge of Y-chromosomal short tandem repeat (Y-STR) mutation rates is essential to determine the most recent common ancestor (MRCA) in familial searching or genealogy research. Up to now, locus-specific mutation rates have been extensively examined especially for commercially available forensic Y-STRs, while haplogroup specific mutation rates have not yet been investigated in detail. Through 450 patrilineally related namesakes distributed over 212 deep-rooting genealogies, the individual mutation rates of 42 Y-STR loci were determined, including 27 forensic Y-STR loci from the Yfiler® Plus kit and 15 additional Y-STR loci (DYS388, DYS426, DYS442, DYS447, DYS454, DYS455, DYS459a/b, DYS549, DYS607, DYS643, DYS724a/b and YCAIIa/b). At least 726 mutations were observed over 148,596 meiosis and individual Y-STR mutation rates varied from 2.83 × 10-4 to 1.86 × 10-2. The mutation rate was significantly correlated with the average allele size, the complexity of the repeat motif sequence and the age of the father. Significant differences in average Y-STR mutations rates were observed when haplogroup 'I & J' (4.03 × 10-3 mutations/generation) was compared to 'R1b' (5.35 × 10-3 mutations/generation) and to the overall mutation rate (5.03 × 10-3 mutations/generation). A difference in allele size distribution was identified as the only cause for these haplogroup specific mutation rates. The haplogroup specific mutation rates were also present within the commercially available Y-STR kits (Yfiler®, PowerPlex® Y23 System and Yfiler® Plus). This observation has consequences for applications where an average Y-STR mutation rate is used, e.g. tMRCA estimations in familial searching and genealogy research.

Genetic-genealogy approach reveals low rate of extrapair paternity in historical Dutch populations.

OBJECTIVE: Evolutionary theory has shown that seeking out extrapair paternity (EPP) can be a viable reproductive strategy for both sexes in pair-bonded species, also in humans. As yet, estimates of the contemporary or historical EPP rate in human population are still rare. In the present study, we estimated the historical EPP rate in the Dutch population over the last 400 years and compared the rate with those obtained for other human populations to determine the evolutionary, cultural, and socio-demographic factors that influence human cuckoldry behavior. METHODS: We estimated the historical EPP rate for the Dutch population via the "genealogical pair method", in which the EPP rate is derived from Y-chromosome mismatches between pairs of individuals that, based on genealogical evidence, share a common paternal ancestor. RESULTS: Based on the analysis of 68 representative genealogical pairs, separated by a total of 1013 fertilization events, we estimated that the historical EPP rate for the Dutch population over the last 400 years was 0.96% per generation (95% confidence interval 0.46%-1.76%). CONCLUSION: The Dutch EPP rate fits perfectly within the range reported for other contemporary and historical populations in Western Europe and was highly congruent with that estimated for neighboring Flanders, despite the socio-economic and religious differences between both populations. The estimated low EPP rate challenges the "dual mating strategy hypothesis" that states that women could obtain fitness benefits by securing investment from one man while cuckolding him to obtain good genes from an affair partner.