Mass Spectrometry Fragmentation Research Articles

Advances in Mass Spectrometry Fragmentation Techniques for Peptide and Protein Characterization: CID to ETD

Mass spectrometry (MS) has transformed the field of proteomics by enabling the precise analysis of proteins and peptides at the molecular level. The heart of this analysis is the process of fragmentation, which plays a pivotal role in elucidating amino acid sequence, identifying post-translational modifications (PTMs), and characterizing structural features. Peptides and proteins are first ionized and then subjected to fragmentation techniques such as Collision-Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), and Electron-Capture Dissociation (ECD). These techniques induce fragmentation along the peptide backbone, generating characteristic fragment ions (b and y ions, c and z ions) that reflect the sequence of amino acids. The mass-to-charge (m/z) ratios of these fragment ions provide crucial data for de novo sequencing and PTM identification. Furthermore, fragmentation patterns can reveal insights into protein folding, interactions, and dynamics. This article provides the detailed view of mass fragmentation techniques in peptides and proteins, highlighting its importance in advancing proteomic research and its applications in fields ranging from biomarker discovery to drug development.

An Equine Protein Atlas Highlights Synovial Fluid Proteome Dynamics during Experimentally LPS-Induced Arthritis.

In human proteomics, substantial efforts are ongoing to leverage large collections of mass spectrometry (MS) fragment ion spectra into extensive spectral libraries (SL) as a resource for data independent acquisition (DIA) analysis. Currently, such initiatives in equine research are still missing. Here we present a large-scale equine SL, comprising 6394 canonical proteins and 89,329 unique peptides, based on data dependent acquisition analysis of 75 tissue and body fluid samples from horses. The SL enabled large-scale DIA-MS based quantification of the same samples to generate a quantitative equine protein distribution atlas to infer dominant proteins in different organs and body fluids. Data mining revealed 163 proteins uniquely identified in a specific type of tissue or body fluid, serving as a starting point to determine tissue-specific or tissue-type-specific proteins. We showcase the SL by highlighting proteome dynamics in equine synovial fluid samples during experimental lipopolysaccharide-induced arthritis. A fuzzy c-means cluster analysis pinpointed SERPINB1, ATRN, NGAL, LTF, MMP1, and LBP as putative biomarkers for joint inflammation. This SL provides an extendable resource for future equine studies employing DIA-MS.

Diastereomer Characterization of PS-Modified Synthetic Oligonucleotides Using Cyclic IMS-MS.

Synthetic oligonucleotides have emerged as effective therapeutics that regulate gene expression to treat and prevent diseases. Oligonucleotide therapeutics are often modified with a substitution of a phosphorothioate (PS) linkage along the phosphodiester backbone to improve the drug performance and stability. The PS modification creates a mixture of diastereomer structures, increasing by a factor of 2n where n is the number of PS linkages. Despite recent draft guidances highlighting the importance of their characterization, analytical methods to measure the resulting diastereomers are currently lacking. Here, we present a method combining tandem mass spectrometry (MS) and tandem ion mobility spectrometry (IMS) using a cyclic IMS-MS instrument to study diastereomers in PS-modified oligonucleotides. This approach requires no enzymatic digestion as the intact oligonucleotides are directly injected into the MS instrument. Analogous to top-down proteomics, MS fragmentation of the intact oligonucleotide results in 3' and 5' fragment ends that have fewer diastereomers than their intact counterpart. Tandem IMS allows for mobility resolution of the diastereomers at the terminal ends. We tested four model oligonucleotides that differ in either the number of PS bonds or sequence to demonstrate the capability of this method to elucidate diastereomer structures on modified oligonucleotides.

Bottom-up Histone Post-translational Modification Analysis using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Tandem Mass Spectrometry.

The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K0) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me1-3) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

Complex Sequence-Defined Heteropolymers Enable Controlled Film Growth in Layer-By-Layer Assembly.

Digitally-encoded poly(phosphodiesters) (d-PPDE) with highly complex primary structures are evaluated for layer-by-layer (LbL) assembly. To be easily decoded by mass spectrometry (MS), these digital polymers contain many different monomers: 2 coding units allowing binary encryption, 1 cleavable spacer allowing controlled MS fragmentation, and 3 mass tags allowing fragment identification. These complex heteropolymers are therefore composed of 6 different motifs. Despite this strong sequence heterogeneity, it is found that they enable a highly controlled LbL film formation. For instance, a regular growth is observed when alternating the deposition of negatively-charged d-PPDE and positively-charged poly(allyl amine hydrochloride) (PAH). Yet, in this approach, the interdistance between consecutive coded d-PPDE layers remains relatively small, which may be an issue for data storage applications, especially for the selective decoding of the stored information. Using poly(sodium 4-styrene sulfonate) (PSS) as an intermediate non-coded polyanion, it is shown that a controlled interdistance between d-PPDE layers can be easily achieved, while still maintaining a regular LbL growth. Last but not least, it is found in this work that d-PPDE of relatively small molecular weight (i.e., significantly smaller than those of PAH and PSS) still enables a controlled LbL assembly.

Alkaloid profiling of the new species Corydalis huangshanensis and other 13 medicinal plants in genus Corydalis.

Corydalis DC., the largest genus of Papaveraceae, comprises numerous species known for their abundant alkaloid content and historical use in clinical medicine. Recently, a new species of genus Corydalis named Corydalis huangshanensis Lu Q. Huang & H. S. Peng was discovered in the Huangshan Mountains of Anhui Province, China. To compare the chemical characteristics of C. huangshanensis and other 13 Corydalis species, aiming to elucidate the potential medicinal value of this new species. The chemical constituents of C. huangshanensis and other 13 medicinal plants of genus Corydalis were analyzed using ultra-high-performance liquid chromatography Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Q-Orbitrap) mass technology. The differences in the alkaloids in the 14 species were distinguished by chemometrics. The mass spectrometry fragmentation information and relative content of 72 alkaloids were obtained. Orthogonal partial least squares discriminant analysis (OPLS-DA) and cluster heat mapping analysis showed that these 14 species were divided into two groups. The clustering relationship between C. huangshanensis and C. decumbens (Thunb.) Pers. was similar, exhibiting similar chemical compositions and characteristics. These results indicate the potential pharmacological effects of C. huangshanensis. This study enhances our understanding of the chemical classification of Corydalis and provides a basis for speculations on the medicinal value of C. huangshanensis.

Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.

Discovery and Identification of Three Homocysteine Metabolites by Chemical Derivatization and Mass Spectrometry Fragmentation.

Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.

Comprehensive identification strategy for rapid profiling of chemical constituents using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry with Rhubarb as an example

In this study, the collision induced dissociation tandem mass spectrometry (CID-MS/MS) fragmentation pathway of chemical components in rhubarb was wholly explored using 34 standards by UHPLC-QTOF-MS/MS in negative ion mode. In consequently, the diagnostic product ions for speedy screening and categorization of chemical components in rhubarb were ascertained based on their MS/MS splitting decomposition patterns and intensity analysis. According to these findings, a fresh two-step data mining strategy had set up. The initial key step involves the use of characteristic product ions and neutral loss to screen for different types of substituents and basic skeletons of compounds. The subsequent key step is to screen and classify different types of compounds based on their characteristic product ions. This method can be utilized for rapid research, classification, and identification of compounds in rhubarb. A total of 356 compounds were rapidly identified or tentatively characterized in three rhubarb species extracts, including 150 acylglucoside, 125 anthraquinone, 65 flavanols and 15 other compounds. This study manifests that the analytical strategy is feasible for the analysis of complex natural products in rhubarb.

Rapid chemical characterization and pharmacological mechanism of Fining Granules in the treatment of chronic bronchitis based on UHPLC–Q-exactive orbitrap mass spectrometer and network pharmacology

BackgroundSenecio cannabifolius Less. is a perennial herb belonging to the Compositae family that has been used in traditional medicine as an antitussive and expectorant for treating chronic bronchitis and acute respiratory infections. Traditionally, Feining Granules are prepared from water extracts of the raw plant material. However, the chemical composition and pharmacological mechanisms of Feining Granules have not been thoroughly investigated. MethodsA systematic strategy for the rapid detection and identification of the constituents of Feining Granules was developed using ultrahigh-performance liquid chromatography–quadrupole-exactive orbitrap mass spectrometry (MS) with parallel reaction monitoring. ResultsOverall, 162 compounds, including flavonoids, alkaloids, organic acids, and others, were identified unambiguously and tentatively by comparing the retention times and MS fragmentation with reference standards and literature data. Ninety-nine of these were reported for the first time to the best of our knowledge. Network pharmacology suggests that Feining Granules can be used to treat chronic bronchitis as they contain active components associated with the ALB, VEGFA, and SRC target genes influenced by HIF-1, VEGF, and other signaling pathways. ConclusionThese results provide information that can help understand the effective substances of S. cannabifolius Less. and improve quality control.

Comprehensive O-Glycan Analysis by Porous Graphitized Carbon Nanoliquid Chromatography-Mass Spectrometry.

The diverse and unpredictable structures of O-GalNAc-type protein glycosylation present a challenge for its structural and functional characterization in a biological system. Porous graphitized carbon (PGC) liquid chromatography (LC) coupled to mass spectrometry (MS) has become one of the most powerful methods for the global analysis of glycans in complex biological samples, mainly due to the extensive chromatographic separation of (isomeric) glycan structures and the information delivered by collision induced fragmentation in negative mode MS for structural elucidation. However, current PGC-based methodologies fail to detect the smaller glycan species consisting of one or two monosaccharides, such as the Tn (single GalNAc) antigen, which is broadly implicated in cancer biology. This limitation is caused by the loss of small saccharides during sample preparation and LC. Here, we improved the conventional PGC nano-LC-MS/MS-based strategy for O-glycan analysis, enabling the detection of truncated O-glycan species and improving isomer separation. This was achieved by the implementation of 2.7 μm PGC particles in both the trap and analytical LC columns, which provided an enhanced binding capacity and isomer separation for O-glycans. Furthermore, a novel mixed-mode PGC-boronic acid-solid phase extraction during sample preparation was established to purify a broad range of glycans in an unbiased manner, including the previously missed mono- and disaccharides. Taken together, the optimized PGC nano-LC-MS/MS platform presents a powerful component of the toolbox for comprehensive O-glycan characterization.

Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation.

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m/z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.

Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.

In-Electrospray source Hydrogen/Deuterium exchange coupled to multistage fragmentation for the investigation of the protonation and fragmentation pathways of gas phase ions.

Identification of molecules in complex natural matrices relies on matching the fragmentation spectra of ions under investigation and the spectra acquired for the corresponding analytical standards. Currently, there are many databases of experimentally measured tandem mass spectrometry spectra (such as NIST, MzCloud, and Metlin), and considerable progress has been made in the development of software for predicting tandem mass spectrometry fragments in silico using combinatorial, machine learning, and quantum chemistry approaches (such as MetFrag, CFM-ID, and QCxMS). However, the electrospray ionization molecules can be ionized at different sites (protonated or deprotonated), and the fragmentation spectra of such ions are different. Here, we are using the combination of the in-ESI source hydrogen/deuterium exchange reaction and MSn fragmentation for the investigation of the fragmentation pathways for different protomers of organic molecules. It is shown that the distribution of the deuterium in the fragment ions reflects the presence of different protomers. For several molecules, the distribution of deuterium was traced up to the MS5 level of fragmentation revealing many unusual and unexpected effects. For example, we investigated the loss of HF from the ciprofloxacin and norfloxacin ions and observed that for ions protonated at -COOH group, the eliminating hydrogen always comes from -NH group. When ions are protonated at another site, the elimination of hydrogen with a probability of 30% occurs from the -NH group, and with a probability of 70%, it originates from other sites on the molecule. Such effects were not described previously. Quantum chemical simulation was used for the verification of the protonated structures and simulation of the corresponding fragmentation spectra.

Forecasting vaping health risks through neural network model prediction of flavour pyrolysis reactions

Vaping involves the heating of chemical solutions (e-liquids) to high temperatures prior to lung inhalation. A risk exists that these chemicals undergo thermal decomposition to new chemical entities, the composition and health implications of which are largely unknown. To address this concern, a graph-convolutional neural network (NN) model was used to predict pyrolysis reactivity of 180 e-liquid chemical flavours. The output of this supervised machine learning approach was a dataset of probability ranked pyrolysis transformations and their associated 7307 products. To refine this dataset, the molecular weight of each NN predicted product was automatically correlated with experimental mass spectrometry (MS) fragmentation data for each flavour chemical. This blending of deep learning methods with experimental MS data identified 1169 molecular weight matches that prioritized these compounds for further analysis. The average number of discrete matches per flavour between NN predictions and MS fragmentation was 6.4 with 92.8% of flavours having at least one match. Globally harmonized system classifications for NN/MS matches were extracted from PubChem, revealing that 127 acute toxic, 153 health hazard and 225 irritant classifications were predicted. This approach may reveal the longer-term health risks of vaping in advance of clinical diseases emerging in the general population.

SpectiCal: m/z Calibration of MS2 Peptide Spectra Using Known Low Mass Ions.

Most tandem mass spectrometry fragmentation spectra have small calibration errors that can lead to suboptimal interpretation and annotation. We developed SpectiCal, a software tool that can read mzML files from data-dependent acquisition proteomics experiments in parallel, compute m/z calibrations for each file prior to identification analysis based on known low-mass ions, and produce information about frequently observed peaks and their explanations. Using calibration coefficients, the data can be corrected to generate new calibrated mzML files. SpectiCal was tested using five public data sets, creating a table of commonly observed low-mass ions and their identifications. Information about the calibration and individual peaks is written in PDF and TSV files. This includes information for each peak, such as the number of runs in which it appears, the percentage of spectra in which it appears, and a plot of the aggregated region surrounding each peak. SpectiCal can be used to compute MS run calibrations, examine MS runs for artifacts that might hinder downstream analysis, and generate tables of detected low-mass ions for further analysis. SpectiCal is freely available at https://github.com/PlantProteomes/SpectiCal.

Tracking Molecular Fragmentation in Electron-Ionization Mass Spectrometry with Ultrafast Time Resolution.

ConspectusMass spectrometry is a powerful analytical method capable of identifying compounds given a minute amount of material. The fragmentation pattern that results following molecular activation serves as a fingerprint that can be matched to a database compound for identification. Over the past half century, studies have addressed and, in many cases, named the chemical reactions that lead to some of the principal fragment ions. Theories have been developed to predict the observed fragmentation patterns, many of which assume that energy redistributes prior to dissociation. However, the existence of rearrangements and nonergodic processes complicates the prediction of fragmentation patterns and the identification of compounds that have yet to be entered into a curated database. To date, very few studies have addressed the time-dependent nature of the fragmentation of radical cations and, in particular, processes occurring with picosecond or shorter time scales where one expects to find nonergodic reactions.This Account focuses on a novel approach that enables tracking of molecular fragmentation in electron-ionization mass spectrometry with ultrafast time resolution. The two challenges that have prevented the time-resolved studies following electron ionization are the random impact parameter and moment of ionization of each molecule. In addition, medium-sized molecules can produce fragmentation patterns with tens if not hundreds of product ions. Spectroscopically interrogating all of these ions as a function of time is another major challenge. We describe strong field disruptive probing, a method that ionizes molecules on a femtosecond time scale and allows us to track in time the formation of all fragment ions simultaneously.Molecular fragmentation following ionization can occur on a very wide range of time scales. Metastable ions can survive from nanoseconds to microseconds; reactions that depend on vibrational energy redistribution can take picoseconds to nanoseconds; and direct dissociation processes and some rearrangements can take place in femtoseconds to picoseconds. All of these processes depend on the dynamics that occur during attoseconds and femtoseconds following the ionization process. Following a discussion of these time scales, we provide three examples of fragmentations that have been studied with femtosecond time resolution. Each of these examples include unforeseen reaction dynamics that involve a nonergodic process, highlighting the importance of time resolution in mass spectrometry. Finally, we explore future challenges and unresolved questions in mass spectrometry and, more broadly, in the domain of electron-initiated chemical reactions.

Forensic implications of novel synthesis of cathinone derivatives by Neber and modified Neber rearrangements

Cathinone and its synthetic analogues are known compounds of clandestine interest. Investigation into novel pathways for synthesising cathinone derivatives has potential for forensic analysis and tracking. The known Neber rearrangement of commercially available phenylpropanones that were evaluated yielded amides described herein and was not viable for clandestine synthesis of cathinone derivatives. Whereas the known modified Neber rearrangement of phenylpropanones that were evaluated via stable aziridine salts and subsequent treatment with acid, gave cathinone derivatives described herein in poor to low yields (2–17%). Assessment of the reagents, equipment, and procedures required for the modified Neber rearrangement was considered as only viable for more advanced clandestine operations. An improved understanding of the potential by-product formation from the modified Neber rearrangement was determined by density functional theory (DFT) of hydrazone to azirine to aziridine intermediates and attempted dynamic NMR spectroscopy of a hydrazone described herein. The substantially lower energy of the azirine step compared to the starting hydrazonium salt step of the reaction mechanism implied that the azirine structure was a short-lived intermediate, and unable to be experimentally determined. New mass-spectral fragmentation data of compounds described herein was reported, where differentiation was observed for some individual compounds at the GC-EIMS fragmentation pattern level. From this study, individual mass-spectrometry fragmentation of key compounds evaluated from the modified Neber rearrangement of commercially available phenylpropanones indicates potential for forensic profiling analysis applications.

Advancing Glucose Conjugated Gibberellins Discovery: A Structure-Oriented Screening and Identification Method for Unraveling Gibberellin Metabolites in Plants.

Gibberellins (GAs) play a pivotal role in modulating plant growth and development. Glucose-conjugated gibberellins (Glc-GAs), a prevalent conjugated form of GAs, regulate intracellular GA levels by the coupling and decoupling of glucose groups. However, the diversity of Glc-GAs identified within individual species remains limited, hinting at a multitude of yet undiscovered gibberellin metabolites. This lacuna poses considerable impediments to research efforts dedicated to comprehensively delineating the GA metabolic pathway. In this study, we developed a structure-oriented screening and identification method for Glc-GAs in plant species by employing LC-MS/MS coupled with chemical derivatization. Through the application of chemical derivatization technique, carboxyl groups on Glc-GAs were labeled which effectively enhanced the sensitivity and selectivity of mass spectrometry detection for these compounds. Concurrently, the integration of mass spectrometry fragmentation and chromatographic retention behavior facilitated the efficient screening and identification of potential Glc-GAs. With this strategy, we screened and identified 12 potential Glc-GAs from six plant species. These findings expand the Glc-GA diversity in plants and contribute to understanding GA metabolic pathways.

Atomistic simulations for investigation of substrate and salt effects on lipid in-source fragmentation in secondary ion mass spectrometry: A follow-up study.

In-source fragmentation (ISF) poses a significant challenge in secondary ion mass spectrometry (SIMS). These fragment ions increase the spectral complexity and can lead to incorrect annotation of fragments as intact species. The presence of salt that is ubiquitous in biological samples can influence the fragmentation and ionization of analytes in a significant manner, but their influences on SIMS have not been well characterized. To elucidate the effect of substrates and salt on ISF in SIMS, we have employed experimental SIMS in combination with atomistic simulations of a sphingolipid on a gold surface with various NaCl concentrations as a model system. Our results revealed that a combination of bond dissociation energy and binding energy between N-palmitoyl-sphingomyelin and a gold surface is a good predictor of fragment ion intensities in the absence of salt. However, ion-fragment interactions play a significant role in determining fragment yields in the presence of salt. Additionally, the charge distribution on fragment species may be a major contributor to the varying effects of salt on fragmentation. This study demonstrates that atomistic modeling can help predict ionization potential when salts are present, providing insights for more accurate interpretations of complex biological spectra.