RESUMO
Ultraviolet photodissociation (UVPD) mass spectrometry has gained attention in recent years for its ability to provide high sequence coverage of intact proteins. However, secondary dissociation of fragment ions, in which fragment ions subjected to multiple laser pulses decompose into small products, is a common phenomenon during UVPD that contributes to limited coverage in the midsection of protein sequences. To counter secondary dissociation, a method involving the application of notched waveforms to modulate the trajectories of fragment ions away from the laser beam, termed fragment ion protection (FIP), was previously developed to reduce the probability of secondary dissociation. This, in turn, increased the number of identified large fragment ions. In the present study, FIP was applied to UVPD of large proteins ranging in size from 29 to 55 kDa, enhancing the abundances of large fragment ions. A stepped-FIP strategy was implemented in which UVPD mass spectra were collected using multiple different amplitudes of the FIP waveforms and then the results from the mass spectra were combined. By using stepped-FIP, the number of fragment ions in the midsections of the sequences increased for all proteins. For example, whereas no fragment ions were identified in the middle section of the sequence for glutamate dehydrogenase (55 kDa, 55+ charge state), 10 sequence ions were identified by using UVPD-FIP.
Assuntos
Espectrometria de Massas/métodos , Proteínas , Análise de Sequência de Proteína/métodos , Raios Ultravioleta , Íons , Fotólise , Proteínas/análise , Proteínas/química , Proteínas/efeitos da radiaçãoRESUMO
Ultraviolet photodissociation (UVPD) produces rich and informative fragmentation of intact protein ions, but in the case of high mass proteins (>30 kDa) the spectra are congested with overlapping isotope patterns of highly charged fragment ions. In the most congested regions, many fragments cannot be confidently identified even when high-resolution mass analyzers and modern deconvolution algorithms are used. Gas-phase ion-ion proton transfer reactions (PTR), which reduce the charge states of highly charged ions, can be used to alleviate this congestion and facilitate the identification of additional fragment ions when performed following UVPD. We have developed protocols for sequentially performing PTR on multiple populations of ions generated by UVPD in a way that can be tailored to balance the depth of characterization with speed and throughput. The improvements in sequence coverage and fragment identifications are demonstrated for four proteins ranging in size from 29 to 56 kDa. Sequence coverages up to 80% were achieved for carbonic anhydrase (29 kDa), 50% for aldolase (39 kDa), 46% for enolase (46 kDa), and 27% for glutamate dehydrogenase (56 kDa), and up to 74% sequence coverage was obtained for 25 kDa antibody drug conjugate subunits in online LC-MS experiments.
Assuntos
Enzimas/química , Imunoconjugados/química , Prótons , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida/métodos , Enzimas/efeitos da radiação , Imunoconjugados/efeitos da radiação , Limite de Detecção , Proteólise/efeitos da radiação , Coelhos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/efeitos da radiação , Espectrometria de Massas em Tandem/métodos , Raios UltravioletaRESUMO
The impact of charging methods on the dissociation behavior of intact proteins in low charge states is investigated using HCD and 193 nm UVPD. Low charge states are produced for seven different proteins using the following four different methods: (1) proton transfer reactions of ions in high charge states generated from conventional denaturing solutions; (2) ESI of proteins in solutions of high ionic strength to enhance retention of folded native-like conformations; (3) ESI of proteins in high pH solutions to limit protonation; and (4) ESI of carbamylated proteins. Comparison of sequence coverages, degree of preferential cleavages, and types and distribution of fragment ions reveals a number of differences in the fragmentation patterns depending on the method used to generate the ions. More notable differences in these metrics are observed upon HCD than upon UVPD. The fragmentation caused by HCD is influenced more significantly by the presence/absence of mobile protons, a factor that modulates the degree of preferential cleavages and net sequence coverages. Carbamylation of the lysines and the N-terminus of the proteins alters the proton mobility by reducing the number of proton-sequestering, highly basic sites as evidenced by decreased preferential fragmentation C-terminal to Asp or N-terminal to Pro upon HCD. UVPD is less dependent on the method used to generate the low charge states and favors non-specific fragmentation, an outcome which is important for obtaining high sequence coverage of intact proteins.
Assuntos
Íons/química , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Bovinos , Cavalos , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Concentração Osmolar , Fotólise , Carbamilação de Proteínas , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Prótons , Raios UltravioletaRESUMO
Unraveling disease mechanisms requires a comprehensive understanding of how the interplay between higher-order structure and protein-ligand interactions impacts the function of a given protein. Recent advances in native mass spectrometry (MS) involving multimodal or higher-energy activation methods have allowed direct interrogation of intact protein complexes in the gas phase, allowing analysis of both composition and subunit connectivity. We report a multistage approach combining collisional activation and 193 nm ultraviolet photodissociation (UVPD) to characterize single amino acid variants of the human mitochondrial enzyme branched-chain amino acid transferase 2 (BCAT2), a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Native electrospray ionization confirms that both proteins exist as homodimers. Front-end collisional activation disassembles the dimers into monomeric subunits that are further interrogated using UVPD to yield high sequence coverage of the mutated region. Additionally, holo (ligand-bound) fragment ions resulting from photodissociation reveal that the mutation causes destabilization of the interactions with a bound cofactor. This study demonstrates the unique advantages of implementing UVPD in a multistage MS approach for analyzing intact protein assemblies.
Assuntos
Substituição de Aminoácidos , Espectrometria de Massas/métodos , Antígenos de Histocompatibilidade Menor/química , Proteínas Mitocondriais/química , Proteínas da Gravidez/química , Transaminases/química , Sítios de Ligação , Humanos , Antígenos de Histocompatibilidade Menor/genética , Proteínas Mitocondriais/genética , Mutação , Proteínas da Gravidez/genética , Fosfato de Piridoxal/química , Transaminases/genética , Raios UltravioletaRESUMO
Ultraviolet photodissociation (UVPD) is a nonselective activation method in which both precursor and fragment ions may absorb photons and dissociate. Photoactivation of fragment ions may result in secondary or multiple generations of dissociation, which decreases the signal-to-noise ratio (S/N) of larger fragment ions owing to the prevalent subdivision of the ion current into many smaller, often less informative, fragment ions. Here we report the use of dipolar excitation waveforms to displace fragment ions out of the laser beam path, thus alleviating the extent of secondary dissociation during 193 nm UVPD. This fragment ion protection (FIP) strategy increases S/N of larger fragment ions and improves the sequence coverage obtained for proteins via retaining information deeper into the midsection of protein sequences.
RESUMO
We demonstrate a method for determining the collision cross-sections (CCSs) of protein ions based on the decay rate of the time-domain transient signal from an Orbitrap mass analyzer. Multiply charged ions of ubiquitin, cytochrome c, and myoglobin were generated by electrospray ionization of both denaturing solutions and ones with high salt content to preserve native-like structures. A linear relationship between the pressure in the Orbitrap analyzer and the transient decay rate was established and used to demonstrate that the signal decay is primarily due to ion-neutral collisions for protein ions across the entire working pressure range of the instrument. The CCSs measured in this study were compared with previously published CCS values measured by ion mobility mass spectrometry (IMS), and results from the two methods were found to differ by less than 7% for all charge states known to adopt single gas-phase conformations.
Assuntos
Citocromos c/análise , Mioglobina/análise , Ubiquitina/análise , Animais , Bovinos , Cavalos , Espectrometria de Mobilidade Iônica , Íons , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We evaluate the impact of carbamylation of the primary amines of the side-chains of Lys and the N-termini on the fragmentation of intact protein ions and the chromatographic properties of a mixture of E. coli ribosomal proteins. The fragmentation patterns of the six unmodified and carbamylated proteins obtained by higher energy collision dissociation (HCD) and ultraviolet photodissociation (UVPD) were compared. Carbamylation significantly reduced the total number of protons retained by the protein owing to the conversion of basic primary amines to non-basic carbamates. Carbamylation caused a significant negative impact on fragmentation of the protein by HCD (i.e., reduced sequence coverage and fewer diagnostic fragment ions) consistent with the mobile proton model, which correlates peptide fragmentation with charge distribution and the opportunity for charge-directed pathways. In addition, fragmentation was enhanced near the N- and C-termini upon HCD of carbamylated proteins. For LCMS/MS analysis of E. coli ribosomal proteins, the retention times increased by 16 min on average upon carbamylation, an outcome attributed to the increased hydrophobicity of the proteins after carbamylation. As noted for both the six model proteins and the ribosomal proteins, carbamylation had relatively little impact on the distribution or types of fragment ions product by UVPD, supporting the proposition that the mechanism of UVPD for intact proteins does not reflect the mobile proton model. Graphical Abstract á .
Assuntos
Aminas/química , Fragmentos de Peptídeos/química , Proteínas/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Bovinos , Galinhas , Citocromos c/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Cavalos , Muramidase/química , Fotólise , Carbamilação de Proteínas , Proteômica , Ubiquitina/química , Raios UltravioletaRESUMO
Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.
Assuntos
Anidrase Carbônica I/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Superóxido Dismutase-1/química , Anidrase Carbônica I/metabolismo , Dimerização , Humanos , Ligantes , Fotólise/efeitos da radiação , Mutação Puntual , Desdobramento de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Raios UltravioletaRESUMO
We describe a strategy for de novo peptide sequencing based on matched pairs of tandem mass spectra (MS/MS) obtained by collision induced dissociation (CID) and 351 nm ultraviolet photodissociation (UVPD). Each precursor ion is isolated twice with the mass spectrometer switching between CID and UVPD activation modes to obtain a complementary MS/MS pair. To interpret these paired spectra, we modified the UVnovo de novo sequencing software to automatically learn from and interpret fragmentation spectra, provided a representative set of training data. This machine learning procedure, using random forests, synthesizes information from one or multiple complementary spectra, such as the CID/UVPD pairs, into peptide fragmentation site predictions. In doing so, the burden of fragmentation model definition shifts from programmer to machine and opens up the model parameter space for inclusion of nonobvious features and interactions. This spectral synthesis also serves to transform distinct types of spectra into a common representation for subsequent activation-independent processing steps. Then, independent from precursor activation constraints, UVnovo's de novo sequencing procedure generates and scores sequence candidates for each precursor. We demonstrate the combined experimental and computational approach for de novo sequencing using whole cell E. coli lysate. In benchmarks on the CID/UVPD data, UVnovo assigned correct full-length sequences to 83% of the spectral pairs of doubly charged ions with high-confidence database identifications. Considering only top-ranked de novo predictions, 70% of the pairs were deciphered correctly. This de novo sequencing performance exceeds that of PEAKS and PepNovo on the CID spectra and that of UVnovo on CID or UVPD spectra alone. As presented here, the methods for paired CID/UVPD spectral acquisition and interpretation constitute a powerful workflow for high-throughput and accurate de novo peptide sequencing.
Assuntos
Peptídeos/química , Análise de Sequência de Proteína , Processos Fotoquímicos , Espectrometria de Massas em Tandem , Raios UltravioletaRESUMO
Confident protein identifications derived from high-throughput bottom-up and top-down proteomics workflows depend on acquisition of thousands of tandem mass spectrometry (MS/MS) spectra with adequate signal-to-noise and accurate mass assignments of the fragment ions. Ultraviolet photodissociation (UVPD) using 193 nm photons has proven to be well-suited for activation and fragmentation of peptides and proteins in ion trap mass spectrometers, but the spectral signal-to-noise ratio (S/N) is typically lower than that obtained from collisional activation methods. The lower S/N is attributed to the dispersion of ion current among numerous fragment ion channels (a,b,c,x,y,z ions). In addition, frequently UVPD is performed such that a relatively large population of precursor ions remains undissociated after the UV photoactivation period in order to prevent overdissociation into small uninformative or internal fragment ions. Here we report a method to improve spectral S/N and increase the accuracy of mass assignments of UVPD mass spectra via resonance ejection of undissociated precursor ions after photoactivation. This strategy, termed precursor ejection UVPD or PE-UVPD, allows the ion trap to be filled with more ions prior to UVPD while at the same time alleviating the space charge problems that would otherwise contribute to the skewing of mass assignments and reduction of S/N. Here we report the performance gains by implementation of PE-UVPD for peptide analysis in an ion trap mass spectrometer.
Assuntos
Peptídeos/análise , Proteínas/análise , Raios Ultravioleta , Espectrometria de Massas em TandemRESUMO
The first application of light-emitting diodes (LEDs) for ultraviolet photodissociation (UVPD) mass spectrometry is reported. LEDs provide a compact, low cost light source and have been incorporated directly into the trapping cell of an Orbitrap mass spectrometer. MS/MS efficiencies of over 50 % were obtained using an extended irradiation period, and UVPD was optimized by modulating the ion trapping parameters to maximize the overlap between the ion cloud and the irradiation volume.
RESUMO
The tremendous number of peptides identified in current bottom-up mass spectrometric workflows, although impressive for high-throughput proteomics, results in little selectivity for more targeted applications. We describe a strategy for cysteine-selective proteomics based on a tagging method that installs a S-Se bond in peptides that is cleavable upon 266 nm ultraviolet photodissociation (UVPD). The alkylating reagent, N-(phenylseleno)phthalimide (NPSP), reacts with free thiols in cysteine residues and attaches a chromogenic benzeneselenol (SePh) group. Upon irradiation of tagged peptides with 266 nm photons, the S-Se bond is selectively cleaved, releasing a benzeneselenol moiety corresponding to a neutral loss of 156 Da per cysteine. Herein we demonstrate a new MS/MS scan mode, UVPDnLossCID, which facilitates selective screening of cysteine-containing peptides. A "prescreening" event occurs by activation of the top N peptide ions by 266 nm UVPD. Peptides exhibiting a neutral loss corresponding to one or more SePh groups are reactivated and sequenced by CID. Because of the low frequency of cysteine in the proteome, unique cysteine-containing peptides may serve as surrogates for entire proteins. UVPDnLossCID does not generate as many peptide spectrum matches (PSMs) as conventional bottom-up methods; however, UVPDnLossCID provides far greater selectivity.
Assuntos
Cisteína/química , Peptídeos/química , Selênio/química , Espectrometria de Massas em Tandem , Raios Ultravioleta , Alquilantes/química , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Citocromos c/química , Citocromos c/metabolismo , Fotólise/efeitos da radiação , Ftalimidas/química , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Compostos de Sulfidrila/químicaRESUMO
The lipid A domain of the endotoxic lipopolysaccharide layer of Gram-negative bacteria is comprised of a diglucosamine backbone to which a variable number of variable length fatty acyl chains are anchored. Traditional characterization of these tails and their linkages by nuclear magnetic resonance (NMR) or mass spectrometry is time-consuming and necessitates databases of pre-existing structures for structural assignment. Here, we introduce an automated de novo approach for characterization of lipid A structures that is completely database-independent. A hierarchical decision-tree MS(n) method is used in conjunction with a hybrid activation technique, UVPDCID, to acquire characteristic fragmentation patterns of lipid A variants from a number of Gram-negative bacteria. Structural assignments are derived from integration of key features from three to five spectra and automated interpretation is achieved in minutes without the need for pre-existing information or candidate structures. The utility of this strategy is demonstrated for a mixture of lipid A structures from an enzymatically modified E. coli lipid A variant. A total of 27 lipid A structures were discovered, many of which were isomeric, showcasing the need for a rapid de novo approach to lipid A characterization.
Assuntos
Lipídeo A/química , Raios Ultravioleta , Escherichia coli/química , Espectrometria de Massas , Conformação ProteicaRESUMO
Detailed structural characterization of intact rough-type lipopolysaccharides (R-LPS) was accomplished using a multi-stage mass spectrometry (MS(3)) strategy consisting of collision-induced dissociation (CID) followed by 193 ultraviolet photodissociation (UVPD) implemented on an Orbitrap Fusion mass spectrometer. Complex mixtures of R-LPS from either Escherichia coli or Salmonella enterica were directly infused into the mass spectrometer using static source nanoelectrospray ionization (nanoESI). An initial CID event performed on an R-LPS precursor produced spectra with abundant ions corresponding to the lipid A and core oligosaccharide (OS) substructures. Comparison of CID spectra of R-LPS ions with varying lipid A and core OS structures verifies that lipid A and core OS ions are consistently produced in high abundance. The resulting lipid A and core OS ions were subsequently activated by CID, high-energy collision-induced dissociation (HCD), or UVPD. For both the lipid A and core OS substructures, HCD and UVPD produced highly informative complementary spectra, with UVPD of the core OS producing an extensive array of cross-ring cleavage fragments. Successful discernment of E. coli R-LPS structures with isomeric core structures confirmed the degree to which subtle structural differences could be determined using this method.
Assuntos
Lipopolissacarídeos/análise , Espectrometria de Massas/métodos , Processos Fotoquímicos , Raios Ultravioleta , Escherichia coli/química , Nanotecnologia , Salmonella enterica/químicaRESUMO
We report the implementation of proton transfer reactions (PTR) and ion parking on an Orbitrap mass spectrometer. PTR/ion parking allows charge states of proteins to be focused into a single lower charge state via sequential deprotonation reactions with a proton scavenging reagent, in this case, a nitrogen-containing adduct of fluoranthene. Using PTR and ion parking, we evaluate the charge state dependence of fragmentation of ubiquitin (8.6 kDa), myoglobin (17 kDa), and carbonic anhydrase (29 kDa) upon higher energy collisional dissociation (HCD) or ultraviolet photodissociation (UVPD). UVPD exhibited less charge state dependence, thus yielding more uniform distributions of cleavages along the protein backbone and consequently higher sequence coverage than HCD. HCD resulted in especially prominent cleavages C-terminal to amino acids containing acidic side-chains and N-terminal to proline residues; UVPD did not exhibit preferential cleavage adjacent to acidic residues but did show enhancement next to proline and phenylalanine.
Assuntos
Anidrases Carbônicas/análise , Mioglobina/análise , Prótons , Ubiquitina/análise , Raios Ultravioleta , Anidrases Carbônicas/metabolismo , Íons/química , Espectrometria de Massas , Processos FotoquímicosRESUMO
The growing use of mass spectrometry in the field of structural biology has catalyzed the development of many new strategies to examine intact proteins in the gas phase. Native mass spectrometry methods have further accelerated the need for methods that can manipulate proteins and protein complexes while minimizing disruption of noncovalent interactions critical for stabilizing conformations. Proton-transfer reactions (PTR) in the gas phase offer the ability to effectively modulate the charge states of proteins, allowing decongestion of mass spectra through separation of overlapping species. PTR was combined with ultraviolet photodissociation (UVPD) to probe the degree of structural changes that occur upon charge reduction reactions in the gas phase. For protein complexes myoglobin·heme (17.6 kDa) and dihydrofolate reductase·methotrexate (19.4 kDa), minor changes were found in the fragmentation patterns aside from some enhancement of fragmentation near the N- and C-terminal regions consistent with slight fraying. After finding little perturbation was caused by charge reduction using PTR, homodimeric superoxide dismutase/CuZn (31.4 kDa) was subjected to PTR in order to separate overlapping monomer and dimer species of the protein that were observed at identical m/z values.
Assuntos
Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Bovinos , Cavalos , Mioglobina/química , Oxirredução , Processos Fotoquímicos , Conformação Proteica , Multimerização Proteica , Prótons , Superóxido Dismutase/química , Tetra-Hidrofolato Desidrogenase/química , Raios UltravioletaRESUMO
Electron transfer dissociation (ETD) has been broadly adopted and is now available on a variety of commercial mass spectrometers. Unlike collisional activation techniques, optimal performance of ETD requires considerable user knowledge and input. ETD reaction duration is one key parameter that can greatly influence spectral quality and overall experiment outcome. We describe a calibration routine that determines the correct number of reagent anions necessary to reach a defined ETD reaction rate. Implementation of this automated calibration routine on two hybrid Orbitrap platforms illustrate considerable advantages, namely, increased product ion yield with concomitant reduction in scan rates netting up to 75% more unique peptide identifications in a shotgun experiment. Graphical Abstract á .
Assuntos
Proteômica/métodos , Proteômica/normas , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , Calibragem , Cinética , Peptídeos/análise , Peptídeos/químicaRESUMO
We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD) at 193 nm for analysis of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the number of identified c- and z-type ions observed when compared to UVPD or ETD alone, as well as generating a more balanced distribution of a/x, b/y, and c/z ion types. Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS(3) format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins.
Assuntos
Transporte de Elétrons , Proteínas/química , Raios UltravioletaRESUMO
The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer's disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissociation methods.
Assuntos
Peptidilprolil Isomerase/análise , Proteômica , Raios Ultravioleta , Humanos , Espectrometria de Massas , Modelos Moleculares , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Processos FotoquímicosRESUMO
Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species-specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold-water species. Protocols for other species (e.g., carp, a warm-water species) can be developed based on these procedures as long as the specific considerations are taken into account.