Sample Preparation Protocol Research Articles

Stability of Plasma Protein Composition in Dried Blood Spot during Storage

Dried blood spot (DBS) technology has become a promising utility for the transportation and storage of biological fluids aimed for the subsequent clinical analysis. The basis of the DBS method is the adsorption of the components of a biological sample onto the surface of a membrane carrier, followed by drying. After drying, the molecular components of the biosample (nucleic acids, proteins, and metabolites) can be analyzed using modern omics, immunological, or genomic methods. In this work, we investigated the safety of proteins on a membrane carrier by tryptic components over time and at different temperatures (+4, 0, 25 °C) and storage (0, 7, 14, and 35 days). It was shown that the choice of a protocol for preliminary sample preparation for subsequent analytical molecular measurements affects the quality of the experimental results. The protocol for preliminary preparation of a biosample directly in a membrane carrier is preferable compared to the protocol with an additional stage of elution of molecular components before the sample preparation procedures. It was revealed that the composition of biosamples remains stable at a temperature of −20 and +4 °C for 35 days of storage, and at +25 °C for 14 days.

Facile NMR approach for profiling curcuminoids present in turmeric

Curcumin, together with demethoxycurcumin and bisdemethoxycurcumin as a whole called curcuminoids, is an active phytochemical constituent present in the turmeric. When it comes to their analysis, most will rely on UV–Visible spectroscopy, HPLC and LC-MS methods. Looking to improve productivity, time and simplicity, we are proposing a 1H NMR based approach for curcuminoids analysis and its applications to different geographical regions. In the present work, sample preparation protocol is reported for the simultaneous determination of curcuminoids using 1H NMR. For the quantification of curcuminoids, 6–7 ppm vinylic proton region in the 1H NMR spectrum was used, where acetone was observed as the suitable solvent in terms of curcuminoids solubility and proper resolution of peak. The result shows that curcumin (46.8–59.50%) was major among all varieties, followed by DMC (22.15–27.70%) and BDMC (17.52–30.29%) except in Andhrapradesh variety, where BDMC (30.29%) was more than DMC (22.89%). These studies were further supported by HPLC analysis.

Evolution of the microstructure of unconsolidated geopolymers by thermoporometry

AbstractThis paper studies the evolution of the pore size distribution of a fresh unconsolidated geopolymer paste between one day and a week, using thermoporometry. This was made possible by following a careful protocol for sample preparation and for analysis by differential scanning calorimetry. In contrast with nitrogen gas adsorption, this method quantifies directly the amount of water in pores. It also does not require heat and vacuum drying, thus maintaining the fragile pore structure of the unconsolidated paste. Moreover, it was found that, in a typical metakaolin‐based sodium geopolymer with a 10 to 20 hours workability period, the porosity gradually refines during the first week while the mesoporous volume is cut in half. This is probably due to the fact that the geopolymer network was still actively condensing from the activation solution. Part of the pore water never froze and, from mass balance, this residual water was attributed to the water bound in the hydration shell of the sodium counter ions. Only a minor occurrence of covalently bound protons as silanol groups was observed. The results presented here usefully complement data obtained by conventional techniques at later ages on consolidated geopolymers. It supports the growing body of literature on the structural evolution of geopolymers with time.

Effects of fixatives on histomagnetic evaluation of iron in rodent spleen

Characterizing the iron distribution in tissue sections is important for several pathologies. Iron content in excised tissue is typically analyzed via histochemical stains, which are dependent on sample preparation and staining protocols. In our recent studies, we examined how magnetic properties of iron can also be exploited to characterize iron distribution in tissue sections in a label free manner. To enable a histomagnetic characterization of iron in a wide variety of available tissues, it is important to extend it to samples routinely prepared for histochemical staining, which often involve use of chemical fixatives. In this study, we took a systematic approach to determine differences between unfixed and formalin-fixed murine spleen tissues in histomagnetic characterization of iron. Superconducting quantum interference device (SQUID) magnetometry and magnetic force microscopy (MFM) were used for macro- and micro-scale histomagnetic characterization. Perl’s stain was used for histochemical characterization of ferric (Fe3+) iron on adjacent sections as that used for MFM analysis. While histochemical analysis revealed a substantial difference in the dispersion of the stain between fixed versus unfixed samples, histomagnetic characterization was not dependent on chemical fixation of tissue. The results from this study reveal that histomagnetic characterization of iron is free from staining artifacts which can be present in histochemical analysis.

Application of High-Performance Liquid Chromatography for Simultaneous Determination of Tenofovir and Creatinine in Human Urine and Plasma Samples.

Tenofovir disoproxil fumarate is widely used in the therapy of human immunodeficiency virus and hepatitis B virus; however, a high concentration of the prodrug effects kidney function damage. To control the effectiveness of kidney functions in treated patients, the level of creatinine in the body must be controlled. This work describes a simple, fast, and “plastic-waste” reducing method for the simultaneous determination of tenofovir and creatinine in human urine and plasma. In both assays, only 50 µL of body fluid was required. The tests were carried out by reversed phase high-performance liquid chromatography with UV detection. In urine samples, the limits of detection for tenofovir and creatinine were 4 µg mL−1 and 0.03 µmol mL−1, respectively. In plasma samples, the limits of detection were 0.15 µg mL−1 for tenofovir and 0.0003 µmol mL−1 for creatinine. The method was applied for the determination of tenofovir and creatinine in human urine and plasma samples. The biggest advantage of the elaborated method is the possibility to determine tenofovir and creatinine in one analytical run in both urine and plasma sample collected from HIV and HBV patients. The possibility to reduce the level of laboratory waste in a sample preparation protocol is in the mainstream of a new trend of analytical chemistry which is based on green chemistry.

Immunophenotyping of Baseline Bone Marrow Reveals a Specific Pattern of Immune Cells Associated with Greater Depth and Sustained Response in Newly Diagnosed Patients Randomized to Krd or Ktd Followed By Carfilzomib Maintenance or Control (AGMT MM 02 Study)

Immunophenotyping of Baseline Bone Marrow Reveals a Specific Pattern of Immune Cells Associated with Greater Depth and Sustained Response in Newly Diagnosed Patients Randomized to Krd or Ktd Followed By Carfilzomib Maintenance or Control (AGMT MM 02 Study)

Discovery based high resolution MS/MS analysis for selection of allergen markers in chocolate and broth powder matrices

Peptide marker identification is an important step in development of a mass spectrometry method for multiple allergen detection, since specificity, robustness and sensitivity of the overall analytical method will depend on the reliability of the proteotypic peptides. As part of the development of a multi-analyte reference method, discovery analysis of two incurred food matrices has been undertaken to select the most reliable peptide markers. Six allergenic ingredients (milk, egg, peanut, soybean, hazelnut, and almond) were incurred into either chocolate or broth powder matrix. Different conditions of protein extraction and purification were tested and the tryptic peptide pools were analysed by untargeted high resolution tandem mass spectrometry and the resulting fragmentation spectra were processed via a commercial software for sequence identification. The analysis performed on incurred foods provides both a prototype effective and straightforward sample preparation protocol and delivers reliable peptides to be included in a standardized selected reaction monitoring method.

Harmonized procedures lead to comparable quantification of total oxylipins across laboratories

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns.

Putative Antimicrobial Peptides of the Posterior Salivary Glands from the Cephalopod Octopus vulgaris Revealed by Exploring a Composite Protein Database.

Cephalopods, successful predators, can use a mixture of substances to subdue their prey, becoming interesting sources of bioactive compounds. In addition to neurotoxins and enzymes, the presence of antimicrobial compounds has been reported. Recently, the transcriptome and the whole proteome of the Octopus vulgaris salivary apparatus were released, but the role of some compounds—e.g., histones, antimicrobial peptides (AMPs), and toxins—remains unclear. Herein, we profiled the proteome of the posterior salivary glands (PSGs) of O. vulgaris using two sample preparation protocols combined with a shotgun-proteomics approach. Protein identification was performed against a composite database comprising data from the UniProtKB, all transcriptomes available from the cephalopods’ PSGs, and a comprehensive non-redundant AMPs database. Out of the 10,075 proteins clustered in 1868 protein groups, 90 clusters corresponded to venom protein toxin families. Additionally, we detected putative AMPs clustered with histones previously found as abundant proteins in the saliva of O. vulgaris. Some of these histones, such as H2A and H2B, are involved in systemic inflammatory responses and their antimicrobial effects have been demonstrated. These results not only confirm the production of enzymes and toxins by the O. vulgaris PSGs but also suggest their involvement in the first line of defense against microbes.

Metabolomics Analysis of Viral Therapeutics.

Virotherapy, enabled by recent advances in the transdisciplinary field of biotechnology, has emerged as a powerful tool for use in anticancer treatment, gene therapy, immunotherapy, etc. Examining the effects of viruses and virus-derived immune-modulating therapeutics is of great fundamental and clinical interest. Here we describe a sample preparation protocol for metabolite extraction from virus-infected tissue, in addition to liquid chromatography-mass spectrometry conditions essential for subsequent analysis. This metabolomics approach delivers highly sensitive and specific metabolite information on various biospecimens. Such an approach may be adopted to monitor biological changes in over 30 relevant metabolic pathways in response to viral infection and also viral therapeutics.

Dosimetry in vitro – exploring the sensitivity of deposited dose predictions vs. affinity, polydispersity, freeze-thawing, and analytical methods

Dose-response by in vitro testing is only valid if the fraction of the particle dose that deposits onto adherent cells is known. Modeling tools such as the ‘distorted grid’ (DG) code are common practices to predict that fraction. As another challenge, workflow efficiency depends on parallelized sample preparation, for which freeze-thaw protocols have been explored earlier, but not their implications on dosimetry. Here we assess the sensitivity of the DG code toward freeze-thaw protocols and variations in user-defined parameters, including the estimation of particle-cell affinity and determination of agglomerate size, which we measure by DLS or AUC. We challenge the sensitivity by materials of varying composition, surface functionalization, and size (TiO2, CeO2, BaSO4, 2x Ag, 3x SiO2). We found that the average effective density is robust, but the dose predictions by different approaches varied typically 2-fold and up to 10-fold; this uncertainty translates directly into the uncertainty of no-effect-concentrations. The use of standardized dispersion protocols increases the uncertainty in doses. The choice of a measurement method and minor details of the particle size distribution strongly influence the modeled dosimetry. Uncertainty is high for very well dispersed nanomaterials; since then, the assumed affinity of particles to cells has a decisive influence. Against this background, the modulation of deposited dose by freeze-thaw protocols is a minor factor that can be controlled by aligning the protocols of sample preparation. However, even then, the uncertainty of deposited doses must be considered when comparing the in vitro toxicity of different nanomaterials.

Occurrence of n-Alkanes in Vegetable Oils and Their Analytical Determination.

Vegetable oils contain endogenous linear hydrocarbons, namely n-alkanes, ranging from n-C21 to n-C35, with odd chain lengths prevalent. Different vegetable oils, as well as oils of the same type, but of different variety and provenience, show typical n-alkane patterns, which could be used as a fingerprint to characterize them. In the first part of this review, data on the occurrence of n-alkanes in different vegetable oils (total and predominant n-alkanes) are given, with a focus on obtaining information regarding variety and geographical origin. The second part aims to provide the state of the art on available analytical methods for their determination. In particular, a detailed description of the sample preparation protocols and analytical determination is reported, pointing out the main drawbacks of traditional sample preparation and possible solutions to implement the analysis with the aim to shift toward rapid and solvent-sparing methods.

A comprehensive evaluation of two sample treatment procedures for the determination of emerging and historical halogenated flame retardants in biota

Two different sample preparation protocols for the determination of 37 emerging and historical halogenated flame retardants (HFRs) in marine tissues were compared with regards to extraction recovery, lipid removal efficiency, repeatability, reproducibility, and ability to measure sub-ng g−1 (dry weight) concentrations in marine biota. One method involved a purification step using gel permeation chromatography (GPC) followed by a HPLC fractionation step on a Partisil amino-cyano normal phase (GPC-Partisil procedure) and the other more traditional method was based on sulphuric acid treatment followed by silica column fractionation (H2SO4-silica procedure). The samples were analysed by gas chromatography (GC) and liquid chromatography (LC) tandem mass spectrometry (MS/MS). Sample fractionation in both methods enabled unique sample preparation procedures to isolate the GC from the LC amenable compounds. Both methods could remove > 99% of the lipids which was necessary prior to GC- and LC-MS/MS analyses. The majority of the target compounds (70%) had acceptable recoveries between 60–120% for both methods. However, the sulphuric acid treatment resulted in the degradation of the TBP-AE and the silica column fractionation resulted in the loss of BEH-TEBP and the elution of PBB-Acr and TBBPA-BME in the unsuitable fraction. High recoveries of DBE-DBCH (α+β), EHTBB, BTBPE, BEH-TEBP, and PBB-Acr were attributed to matrix effects, suggesting the need to use isotope-labelled surrogate standards of the target compounds. The optimisation of the silica column chromatography, GPC, and Partisil fractionation is described and discussed to afford easy implementation of the method. The method using GPC followed by Partisil fractionation is more efficient and more reproducible than the sulphuric acid-silica procedure. The application of this method to marine biota reference materials revealed the presence of relatively high concentrations of DBE-DBCH isomers and BDE-47 in fish samples. The method detection limits comply with the recommendations of the European Commission.

Natural alkaloids from narrow-leaf and yellow lupins transfer to soil and soil solution in agricultural fields

BackgroundLupin is a promising legume crop, belongs to the Fabaceae (or Leguminosae) family. Lupin production for traditional and functional foods or animal feed is limited, due to the content of toxic quinolizidine (QA)s and indole alkaloids (IA)s. These compounds may not only pose a risk to humans and animals through food consumption, but may also affect soil and aquatic ecosystems. Field experiments were conducted to study the alkaloids content in both narrow-leaved or blue (L. angustifolius) and yellow (L. luteus) lupin plant tissue during a full growing season and understand the environmental fate of alkaloids in soil and water. Suction cups were used to collect soil pore water (soil solution) at four depths: 10, 25, 50 and 70 cm. A full protocol for sample preparation and UPLC–MS/MS quantification of alkaloids in plant, soil and water was developed.ResultsDuring the field experiments the alkaloids in the plant tissues increased, at the harvest stage the content was highest with 21.4 and 24.6 mg/kg dry weight (dw) for blue and yellow lupin, respectively. In soil, alkaloids quantified during the growing season (max concentration was 1.3 × 102 µg/kg dw) and even detected after harvest (0.2 µg/kg dw). In soil pore water samples, alkaloids were not detected during summer, but the concentrations increased to 9.8 × 102 and 1.5 × 103 ng/L for blue and yellow, respectively, in September when autumn precipitation began.ConclusionsThe results show the amount of alkaloids transferred from plant tissue into soil and soil pore water estimated to be on average 0.016% and 0.005% in soil and soil pore water, respectively. Alkaloids leached from topsoil to subsoil layers; the concentrations decline with soil depth. This study demonstrates that alkaloids are mobile compounds in the soil environments, thus lupin production may affect soil or aquatic ecosystems, and reduce water quality.

Efficient Synthesis of Trifluoromethylated Purine Ribonucleosides and Ribonucleotides.

The protocols presented in this article describe highly detailed synthesis of trifluoromethylated purine nucleotides and nucleosides (G and A). The procedure involves trifluoromethylation of properly protected (acetylated) nucleosides, followed by deprotection leading to key CF3 -containing nucleosides. This gives synthetic access to 8-CF3 -substituted guanosine derivatives and three adenosine derivatives (8-CF3 , 2-CF3 , and 2,8-diCF3 ). In further steps, phosphorylation and phosphate elongation (for selected examples) result in respective trifluoromethylated nucleoside mono-, di-, and triphosphates. Support protocols are included for compound handling, purification procedures, analytical sample preparation, and analytical techniques used throughout the performance of the basic protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of trifluoromethylated guanosine and adenosine derivatives Basic Protocol 2: Synthesis of trifluoromethylated guanosine and adenosine monophosphates Basic Protocol 3: Synthesis of phosphorimidazolides of 8-CF3 GMP and 8-CF3 AMP Basic Protocol 4: Synthesis of trifluoromethylated guanosine and adenosine oligophosphates Support Protocol 1: TLC sample preparation and analysis Support Protocol 2: Purification protocol for Basic Protocol 1 Support Protocol 3: HPLC analysis and preparative HPLC Support Protocol 4: Ion-exchange chromatography.

On sample preparation methods for fermented beverage VOCs profiling by GCxGC-TOFMS

IntroductionAromas and tastes have crucial influences on the quality of fermented beverages. The determination of aromatic compounds requires global non-targeted profiling of the volatile organic compounds (VOCs) in the beverages. However, experimental VOC profiling result depends on the chosen VOC collection method.ObjectivesThis study aims to observe the impact of using different sample preparation techniques [dynamic headspace (DHS), vortex-assisted liquid–liquid microextraction (VALLME), multiple stir bar sorptive extraction (mSBSE), solid phase extraction (SPE), and solid phase micro-extraction (SPME)] to figure out the most suitable sample preparation protocol for profiling the VOCs from fermented beverages.MethodsFive common sample preparation methods were studied with beer, cider, red wine, and white wine samples. After the sample preparation, collected VOCs were analyzed by two-dimensional gas chromatography coupled with time of flight mass spectrometry (GCxGC-TOFMS).ResultsGCxGC oven parameters can be optimized with the Box–Behnken surface response model and response measure on peak dispersion. Due to the unavoidable column and detector saturation during metabolomic analysis, errors may happen during mass spectrum construction. Profiling results obtained with different sample preparation methods show considerable variance. Common findings occupy a small fraction of total annotated VOCs. For known fermentative aromas, best coverage can be reached by using SPME together with SPE for beer, and VALLME for wine and cider.ConclusionsGCxGC-TOFMS is a promising tool for non-targeted profiling on VOCs from fermented beverages. However, a proper data processing protocol is lacking for metabolomic analysis. Each sample preparation method has a specific profiling spectrum on VOC profiling. The coverage of the VOC metabolome can be improved by combining complementary methods.

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.

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20-100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Quantitative determination of isoflavonoids in Ononis species by UPLC-UV-DAD.

The root of the Ononis species has been used internally and externally in ethnomedicine for centuries and contains biologically valuable isoflavonoid compounds. Therefore, it is important to obtain quantitative information about the isoflavonoid profile of these plants. In this article we aimed to develop an optimised sample preparation protocol alongside a validated method for the quantitative measurement of isoflavones, isoflavanones and pterocarpans in the form of glucosides and aglycones, in order to compare the specialised metabolites of Ononis spinosa L. and O. arvensis L. Quantitative determination was carried out by the means of ultra-performance liquid chromatography coupled with ultraviolet diode-array detection (UPLC-UV-DAD). An optimised sample preparation method was developed to transform malonyl glucosides to their glucosidic forms. Chromatographic methods were created for the baseline separation of isoflavones, isoflavanones and pterocarpans alongside with their glucosides. Altogether 12 compounds were evaluated quantitatively in samples of O. spinosa and O. arvensis. As a result, no characteristic change could be observed between the two species regarding their isoflavonoid pattern.

Reconstruction of temperature experienced by Pacific bluefin tuna Thunnus orientalis larvae using SIMS and microvolume CF-IRMS otolith oxygen isotope analyses

This study aimed to reconstruct temperatures experienced during the larval period by adult Pacific bluefin tuna Thunnus orientalis using high-resolution otolith stable oxygen isotope (δ18O) analysis. A novel otolith sample preparation protocol for secondary ion mass spectrometry (SIMS) analysis developed in this study reduced the background noise of SIMS measurements, enabling analyses of >10 times higher resolution around the otolith core compared to previous studies using conventional isotope ratio mass spectrometry (IRMS). The values obtained from SIMS were compared to those obtained by microvolume δ18Ootolith analysis using micromilling and conventional continuous-flow IRMS (CF-IRMS). There was a systematic offset (average 0.41‰ with SIMS resulting in lower values) most likely caused by matrix effects on SIMS δ18Ootolith values that can be calibrated using a strong linear relationship between SIMS and CF-IRMS measurements (r2 = 0.78, p < 0.001). The core-to-edge δ18Ootolith of 5 Pacific bluefin tuna revealed fine-scale seasonal variations in water temperature agreeing with known migration patterns. In addition, the ambient water temperature experienced during larval stages (about 10-20 d post hatch) estimated from otolith core δ18O ranged from 26.7 to 30.7°C, overlapping with temperatures associated with the occurrence of larval Pacific bluefin tuna. Combining SIMS and microvolume CF-IRMS δ18O otolith analyses offers a microscale examination of fish ecology that is not possible with conventional IRMS techniques. This novel method is particularly useful for understanding the early life history of fish that may be affected by climate change and reconstructing a well-resolved migration history for fish species that have small otoliths and/or narrow growth increments.