bims-metlip 2021-07-25 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–07–25
thirteen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Development of an Accurate Mass Retention Time Database for Untargeted Metabolomic Analysis and Its Application to Plasma and Urine Pediatric Samples.
Strategy for Nontargeted Metabolomic Annotation and Quantitation Using a High-Resolution Spectral-Stitching Nanoelectrospray Direct-Infusion Mass Spectrometry with Data-Independent Acquisition.
Bridging the Polar and Hydrophobic Metabolome in Single-Run Untargeted Liquid Chromatography-Mass Spectrometry Dried Blood Spot Metabolomics for Clinical Purposes.
Optimization of Sample Preparation for Metabolomics Exploration of Urine, Feces, Blood and Saliva in Humans Using Combined NMR and UHPLC-HRMS Platforms.
Maternal Rat Metabolomics: Amniotic Fluid and Placental Metabolic Profiling Workflows.
Ultrasensitive and High Reproducible Detection of Urinary Metabolites Using the Tip-Contact Extraction Method Coupled with Negative LDI-MS.
Isocyanate derivatization coupled with phospholipid removal microelution-solid phase extraction for the simultaneous quantification of (S)-metoprolol and (S)-α-hydroxymetoprolol in human plasma with LC-MS/MS.
High-Throughput UHPLC/MS/MS-Based Metabolic Profiling Using a Vacuum Jacketed Column.
Signal Separated Quantification of γ-Hydroxybutyrate With Liquid Chromatography-Tandem Mass Spectrometry in Human Urine and Serum as an Improvement of the Analyte Adduct Ion Based Quantification.
Single Cell Metabolite Detection Using Inertial Microfluidics-Assisted Ion Mobility Mass Spectrometry.
Rapid quantification of fatty acids in plant oils and biological samples by LC-MS.
Cross-Normalization of MALDI Mass Spectrometry Imaging Data Improves Site-to-Site Reproducibility.
Mass spectrometry-based methods for the advanced characterization and structural analysis of lignin: A review.

Molecules. 2021 Jul 13. pii: 4256. [Epub ahead of print]26(14):

Development of an Accurate Mass Retention Time Database for Untargeted Metabolomic Analysis and Its Application to Plasma and Urine Pediatric Samples.

Chiara Lavarello, Sebastiano Barco, Martina Bartolucci, Isabella Panfoli, Emanuele Magi, Gino Tripodi, Andrea Petretto, Giuliana Cangemi.

  Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.

Keywords:  LC-HRMS; chromatography; library; metabolomics; pediatrics

DOI:  https://doi.org/10.3390/molecules26144256
Anal Chem. 2021 Jul 22.

Strategy for Nontargeted Metabolomic Annotation and Quantitation Using a High-Resolution Spectral-Stitching Nanoelectrospray Direct-Infusion Mass Spectrometry with Data-Independent Acquisition.

Lichao Wang, Wangjie Lv, Xiaoshan Sun, Fujian Zheng, Tianrun Xu, Xinyu Liu, Hang Li, Xin Lu, Xiaojun Peng, Chunxiu Hu, Guowang Xu.

Direct-infusion nanoelectrospray ionization high-resolution mass spectrometry (DI-nESI-HRMS) is an alternative approach to chromatography-MS-based techniques for nontargeted metabolomics, offering a high sample throughout. However, its annotation accuracy of analytes is still full of challenges. In this study, we proposed a strategy for the annotation and quantitation of nontargeted metabolomic data using a spectral-stitching DI-nESI-HRMS with data-independent acquisition. The metabolite annotation strategy included the isotopic distribution, MS/MS spectrum similarity, and precursor and product ion correlation as well as matching of the extracted metabolite features along with the targeted metabolite precursors. Two groups of mixed standard solutions containing 40 and 79 metabolites were, respectively, used to establish the metabolite annotation strategy and validate its reliability. The results showed that the detected standards could be well annotated at top three explanations and total qualitative percentages were 100% (40 of 40) for the standard solution and 94.9% (74 of 78) for the standards spiked into the serum matrix. The intensity of the precursor ions was used for quantitation except for isomers, which were quantified by the intensities of the characteristic product ions if available. Finally, the strategy was applied to study serum metabolomics in diabetes, and the results demonstrated that it is promising for a large-scale cohort metabolomic study.

DOI: https://doi.org/10.1021/acs.analchem.1c01480
J Proteome Res. 2021 Jul 23.

Bridging the Polar and Hydrophobic Metabolome in Single-Run Untargeted Liquid Chromatography-Mass Spectrometry Dried Blood Spot Metabolomics for Clinical Purposes.

Hanne Bendiksen Skogvold, Elise Mørk Sandås, Anja Østeby, Camilla Løkken, Helge Rootwelt, Per Ola Rønning, Steven Ray Wilson, Katja Benedikte Prestø Elgstøen.

  Dried blood spot (DBS) metabolite analysis is a central tool for the clinic, e.g., newborn screening. Instead of applying multiple analytical methods, a single liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolites spanning from highly polar glucose to hydrophobic long-chain acylcarnitines. For liquid chromatography, a diphenyl column and a multi-linear solvent gradient operated at elevated flow rates allowed for an even-spread resolution of diverse metabolites. Injecting moderate volumes of DBS organic extracts directly, in contrast to evaporation and reconstitution, provided substantial increases in analyte recovery. Q Exactive MS settings were also tailored for sensitivity increases, and the method allowed for analyte retention time and peak area repeatabilities of 0.1-0.4 and 2-10%, respectively, for a wide polarity range of metabolites (log P -4.4 to 8.8). The method's performance was suited for both untargeted analysis and targeted approaches evaluated in clinically relevant experiments.

Keywords:  LC−MS; dried blood spots; inborn errors of metabolism; metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.1c00326
Molecules. 2021 Jul 06. pii: 4111. [Epub ahead of print]26(14):

Optimization of Sample Preparation for Metabolomics Exploration of Urine, Feces, Blood and Saliva in Humans Using Combined NMR and UHPLC-HRMS Platforms.

Cécile Martias, Nadine Baroukh, Sylvie Mavel, Hélène Blasco, Antoine Lefèvre, Léa Roch, Frédéric Montigny, Julie Gatien, Laurent Schibler, Diane Dufour-Rainfray, Lydie Nadal-Desbarats, Patrick Emond.

  Currently, most clinical studies in metabolomics only consider a single type of sample such as urine, plasma, or feces and use a single analytical platform, either NMR or MS. Although some studies have already investigated metabolomics data from multiple fluids, the information is limited to a unique analytical platform. On the other hand, clinical studies investigating the human metabolome that combine multi-analytical platforms have focused on a single biofluid. Combining data from multiple sample types for one patient using a multimodal analytical approach (NMR and MS) should extend the metabolome coverage. Pre-analytical and analytical phases are time consuming. These steps need to be improved in order to move into clinical studies that deal with a large number of patient samples. Our study describes a standard operating procedure for biological specimens (urine, blood, saliva, and feces) using multiple platforms (1H-NMR, RP-UHPLC-MS, and HILIC-UHPLC-MS). Each sample type follows a unique sample preparation procedure for analysis on a multi-platform basis. Our method was evaluated for its robustness and was able to generate a representative metabolic map.

Keywords:  1H-NMR; UHPLC-HRMS; biological fluids; extraction protocol; metabolomic card

DOI:  https://doi.org/10.3390/molecules26144111
J Proteome Res. 2021 Jul 20.

Maternal Rat Metabolomics: Amniotic Fluid and Placental Metabolic Profiling Workflows.

Alexandra Bourdin-Pintueles, Laurent Galineau, Lydie Nadal-Desbarats, Camille Dupuy, Sylvie Bodard, Julie Busson, Antoine Lefèvre, Patrick Emond, Sylvie Mavel.

  Studying the metabolome of specific gestational compartments is of growing interest in the context of fetus developmental disorders. However, the metabolomes of the placenta and amniotic fluid (AF) are poorly characterized. Therefore, we present the validation of a fingerprinting methodology. Using pregnant rats, we performed exhaustive and robust extractions of metabolites in the AF and lipids and more polar metabolites in the placenta. For the AF, we compared the extraction capabilities of methanol (MeOH), acetonitrile (ACN), and a mixture of both. For the placenta, we compared (i) the extraction capabilities of dichloromethane, methyl t-butyl ether (MTBE), and butanol, along with (ii) the impact of lyophilization of the placental tissue. Analyses were performed on a C18 and hydrophilic interaction liquid chromatography combined with high-resolution mass spectrometry. The efficiency and the robustness of the extractions were compared based on the number of the features or metabolites (for untargeted or targeted approach, respectively), their mean total intensity, and their coefficient of variation (% CV). The extraction capabilities of MeOH and ACN on the AF metabolome were equivalent. Lyophilization also had no significant impact and usefulness on the placental tissue metabolome profiling. Considering the placental lipidome, MTBE extraction was more informative because it allowed extraction of a slightly higher number of lipids, in higher concentration. This proof-of-concept study assessing the metabolomics and lipidomics of the AF and the placenta revealed changes in both metabolisms, at two different stages of rat gestation, and allowed a detailed prenatal metabolic fingerprinting.

Keywords:  LC-MS; NMR; fingerprinting methodology; lipidomics; pregnant rodent; targeted; untargeted; validation

DOI:  https://doi.org/10.1021/acs.jproteome.1c00145
J Proteome Res. 2021 Jul 19.

Ultrasensitive and High Reproducible Detection of Urinary Metabolites Using the Tip-Contact Extraction Method Coupled with Negative LDI-MS.

Xinrong Jiang, Fengbin Gao, Xiaoming Chen, Yanlan Yu, Guoqing Ding, Jianmin Wu.

  More and more evidence has proved that urinary metabolites can instantly reflect disease state. Therefore, ultra-sensitive and reproducible detection of urinary metabolites in a high-throughput way is urgently desirable for clinical diagnosis. Matrix-free laser desorption/ionization mass spectrometry (LDI-MS) is a high-throughput platform for metabolites detection, but it is encountered by severe interference from numerous salts in urine samples, because the crystallized urine salt on dried samples could result in poor reproducibility in LDI-MS detection. The present work proposed a tip-contact extraction (TCE) technique to eliminate interference from the urine salt. Vertical silicon nanowire arrays decorated with the fluorinated ethylene propylene film (FEP@VSiNWs) could effectively extract metabolites from the urine sample dropping on its surface. High salt tolerance was observed in the subsequent LDI-MS detection of the metabolites extracted on the tip of FEP@VSiNWs even in the presence of 1 M urea. Stable and reproducible mass spectra for non-target metabolic analysis were obtained in real urine samples with different dilution folds. Urinary metabolites collected from bladder cancer (BC) patients were reliably profiled by the TCE method coupled with negative LDI-MS. Based on this platform, potential metabolic biomarkers that can distinguish BC patients and normal controls were uncovered.

Keywords:  laser desorption ionization; metabolite fingerprinting; non-invasive bladder cancer diagnosis; salt tolerance; silicon nanowires; tip−contact extraction; urine metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.1c00340
J Pharm Biomed Anal. 2021 Jul 13. pii: S0731-7085(21)00374-5. [Epub ahead of print]204 114263

Isocyanate derivatization coupled with phospholipid removal microelution-solid phase extraction for the simultaneous quantification of (S)-metoprolol and (S)-α-hydroxymetoprolol in human plasma with LC-MS/MS.

Maxime Meloche, Martin Jutras, Isabelle St-Jean, Simon de Denus, Grégoire Leclair.

  A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the quantification of (S)-metoprolol (MET) and its main metabolite, (S)-α-hydroxymetoprolol (OH-MET). Human plasma samples (50 μL) were spiked with both analytes and their deuterated internal standards (IS) (S)-MET-d7 and α-OH-MET-d5. Phospholipid removal microelution-solid phase extraction (PRM-SPE) was performed using a 4-step protocol with Oasis PRiME MCX μElution 96-well cartridges. The eluates were reconstituted in 100 μL of acetonitrile with 50 μg/mL (S)-α-methylbenzyl isocyanate (MBIC) for chiral derivatization. After 60 min at room temperature, the reaction was quenched using 100 μL of water 2 % formic acid. Chromatographic separation of the derivatized analytes was performed on a Kinetex phenyl-hexyl core-shell stationary phase with an elution gradient. Mobile phases were composed of a mixture of water and methanol, with ammonium formate and formic acid as buffers. Total runtime was 15 min. Analyte detection was performed by an AB/SCIEX 4000 QTRAP mass spectrometer with multiple reaction monitoring. Chromatograms showed MBIC successfully reacted with racemic MET, α-OH-MET, and their respective IS. Detection by positive electrospray ionization did not reveal derivatized by-products. Quantification ranges were validated for (S)-MET and (S)-α-OH-MET between 0.5-500 and 1.25-500 ng/mL, respectively, with correlation coefficients (r2) >0.9906. The PRM-SPE assay showed low matrix effects (86.9-104.0 %) and reproducible recoveries (69.4-78.7 %) at low, medium, and high quality control (QC) levels. Precision and accuracy were all comprised between 85-115 % for all three QCs, and between 80-120 % for the lower limit of quantification, for intra- and inter-day values (n = 6, 3 consecutive days). Non-derivatized analytes were stable at room temperature, after 3 freeze-thaw cycles, and stored for 30 days at -80 °C (n = 4). Reinjection reproducibility of a previously validated batch was achieved after 8 days under auto-sampler conditions, indicating the stability of (S)-MET and (S)-α-OH-MET derivatives. Its clinical use was established in a cohort of 50 patients and could be used to further investigate the clinical impact of (S)-MET concentrations.

Keywords:  Chiral assay; Isocyanate derivatization; LC–MS/MS; Metoprolol; Phospholipid removal microelution-solid phase extraction

DOI:  https://doi.org/10.1016/j.jpba.2021.114263
Anal Chem. 2021 Jul 19.

High-Throughput UHPLC/MS/MS-Based Metabolic Profiling Using a Vacuum Jacketed Column.

Robert S Plumb, Thomas McDonald, Paul D Rainville, Jason Hill, Lee A Gethings, Kelly A Johnson, Ian D Wilson.

In UHPLC, frictional heating from the eluent flowing through the column at pressures of ca. 10-15 Kpsi causes radial diffusion via temperature differences between the center of the column and its walls. Longitudinal dispersion also occurs due to temperature gradients between the inlet and outlet. These effects cause band broadening but can be mitigated via a combination of vacuum jacketed stainless steel tubing, reduced column end nut mass, and a constant temperature in the column from heating the inlet fitting. Here, vacuum jacketed column (VJC) technology, employing a novel column housing located on the source of the mass spectrometer and minimized tubing from the column outlet to the electrospray probe, was applied to profiling metabolites in urine. For a 75 s reversed-phase gradient separation, the average peak widths for endogenous compounds in urine were 1.2 and 0.6 s for conventional LC/MS and VJC systems, respectively. The peak tailing factor was reduced from 1.25 to 1.13 when using the VJC system compared to conventional UHPLC, and the peak capacity increased from 65 to 120, with a 25% increase in features detected in urine. The increased resolving power of the VJC system reduced co-elution, simplifying MS and MS/MS spectra, providing a more confident metabolite identification. The increased LC performance also gave more intense MS peaks, with a 10-120% increase in response, improving the quality of the MS data and detection limits. Reducing the LC gradient duration to 37 s gave peak widths of ca. 0.4 s and a peak capacity of 84.

DOI: https://doi.org/10.1021/acs.analchem.1c01982
J Anal Toxicol. 2021 Jul 20. pii: bkab083. [Epub ahead of print]

Signal Separated Quantification of γ-Hydroxybutyrate With Liquid Chromatography-Tandem Mass Spectrometry in Human Urine and Serum as an Improvement of the Analyte Adduct Ion Based Quantification.

Marek Dziadosz, Michael Klintschar, Jörg Teske.

  The aim of the work was the development and validation of an LC-MS/MS γ-hydroxybutyrate (GHB) quantification method in urine and human serum by the use of the analyte adduct ion formation strategy. A combined detection with a conventional precursor ion in the negative electrospray mode and additionally GHB adduct ions with both sodium acetate and lithium acetate was in focus. Therefore GHB quantification based on separated MS/MS signals. Two tandem mass spectrometers representing different MS/MS generations (Sciex API 4000 QTrap and Sciex API 5500 QTrap) were used for method validation and comparison. Shimadzu HPLC Systems equipped with a Luna 5 µm C18 (2) 100 A, 150 mm × 2 mm analytical column were applied successfully for sample analyses. Infusion experiments were performed for adduct identification and analyte detection optimisation. Sample preparation could be limited to a simple and fast protein precipitation/sample dilution. An effective signal separated GHB quantification with three independent precursor ions representing separated areas of the mass spectrum was developed, validated according to forensic guidelines and applied in the routine. The developed and applied strategy resulted in a higher safety factor for the analyte quantification performed in the forensic toxicology. A relevant analytical improvement could be achieved with this alternative adduct based GHB analysis since a good correlation of analyte concentrations calculated on the basis of separated signals was stated as useful analytical information.

Keywords:  Adduct formation; Adduct fragmentation; GHB; Signal separation; γ-hydroxybutyrate

DOI:  https://doi.org/10.1093/jat/bkab083
Anal Chem. 2021 Jul 22.

Single Cell Metabolite Detection Using Inertial Microfluidics-Assisted Ion Mobility Mass Spectrometry.

Leicheng Zhang, Tengfei Xu, Jingtao Zhang, Stephen Choong Chee Wong, Mark Ritchie, Han Wei Hou, Yulan Wang.

Single-cell metabolite measurement remains highly challenging due to difficulties related to single cell isolation, metabolite detection, and identification of low levels of metabolites. Here, as a first step of the technological development, we propose a novel strategy integrating spiral inertial microfluidics and ion mobility mass spectrometry (IM-MS) for single-cell metabolite detection and identification. Cells in methanol suspension are inertially focused into a single stream in the spiral microchannel. This stream of separated cells is delivered to the nanoelectrospray needle to be lysed and ionized and subsequently analyzed in real time by IM-MS. This analytical system enables six to eight single-cell metabolic fingerprints to be collected per minute, including gas-phase collisional cross section (CCS) measurements as an additional molecular descriptor, giving increased confidence in metabolite identification. As a proof of concept, the metabolic profiles of three types of cancer cells (U2OS, HepG2, and HepG2.215) were successfully screened, and 19 distinct lipids species were identified with CCS value filtering. Furthermore, principal component analysis (PCA) showed differentiation of the three cancer cell lines, mainly due to cellular surface phospholipids. Taken together, our technology platform offers a simple and efficient method for single-cell lipid profiling, with additional ion mobility separation of lipids significantly improving the confidence toward identification of metabolites.

DOI: https://doi.org/10.1021/acs.analchem.1c00106
Anal Bioanal Chem. 2021 Jul 22.

Rapid quantification of fatty acids in plant oils and biological samples by LC-MS.

Elisabeth Koch, Michelle Wiebel, Carolin Hopmann, Nadja Kampschulte, Nils Helge Schebb.

  Analysis of fatty acids (FA) in food and biological samples such as blood is indispensable in modern life sciences. We developed a rapid, sensitive and comprehensive method for the quantification of 41 saturated and unsaturated fatty acids by means of LC-MS. Optimized chromatographic separation of isobaric analytes was carried out on a C8 reversed phase analytical column (100 × 2.1 mm, 2.6 μm core-shell particle) with a total run time of 15 min with back pressure lower than 300 bar. On an old triple quadrupole instrument (3200, AB Sciex), pseudo selected reaction monitoring mode was used for quantification of the poorly fragmenting FA, yielding limits of detection of 5-100 nM. Sample preparation was carried out by removal of phospholipids and triglycerides by solid-phase extraction (non-esterified fatty acids in oils) or saponification in iso-propanol (fatty acyls). This is not only a rapid strategy for quantification of fatty acyls, but allows the direct combination with the LC-MS-based analysis of fatty acid oxidation products (eicosanoids and other oxylipins) from the same sample. The concentrations of fatty acyls determined by means of LC-MS were consistent with those from GC-FID analysis demonstrating the accuracy of the developed method. Moreover, the method shows high precisions with a low intra-day (≤ 10% for almost all fatty acids in plasma and ≤ 15% in oils) and inter-day as well as inter-operator variability (< 20%). The method was successfully applied on human plasma and edible oils. The possibility to quantify non-esterified fatty acids in samples containing an excess of triacylglycerols and phospholipids is a major strength of the described approach allowing to gain new insights in the composition of biological samples.

Keywords:  Chromatographic separation; Non-esterified fatty acids; Oxylipins; Pseudo-SRM; Saponification

DOI:  https://doi.org/10.1007/s00216-021-03525-y
Anal Chem. 2021 Jul 23.

Cross-Normalization of MALDI Mass Spectrometry Imaging Data Improves Site-to-Site Reproducibility.

Tobias Boskamp, Rita Casadonte, Lena Hauberg-Lotte, Sören Deininger, Jörg Kriegsmann, Peter Maass.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is an established tool for the investigation of formalin-fixed paraffin-embedded (FFPE) tissue samples and shows a high potential for applications in clinical research and histopathological tissue classification. However, the applicability of this method to serial clinical and pharmacological studies is often hampered by inevitable technical variation and limited reproducibility. We present a novel spectral cross-normalization algorithm that differs from the existing normalization methods in two aspects: (a) it is based on estimating the full statistical distribution of spectral intensities and (b) it involves applying a non-linear, mass-dependent intensity transformation to align this distribution with a reference distribution. This method is combined with a model-driven resampling step that is specifically designed for data from MALDI imaging of tryptic peptides. This method was performed on two sets of tissue samples: a single human teratoma sample and a collection of five tissue microarrays (TMAs) of breast and ovarian tumor tissue samples (N = 241 patients). The MALDI MSI data was acquired in two labs using multiple protocols, allowing us to investigate different inter-lab and cross-protocol scenarios, thus covering a wide range of technical variations. Our results suggest that the proposed cross-normalization significantly reduces such batch effects not only in inter-sample and inter-lab comparisons but also in cross-protocol scenarios. This demonstrates the feasibility of cross-normalization and joint data analysis even under conditions where preparation and acquisition protocols themselves are subject to variation.

DOI: https://doi.org/10.1021/acs.analchem.1c01792
Mass Spectrom Rev. 2021 Jul 22.

Mass spectrometry-based methods for the advanced characterization and structural analysis of lignin: A review.

Dane R Letourneau, Dietrich A Volmer.

  Lignin is currently one of the most promising biologically derived resources, due to its abundance and application in biofuels, materials and conversion to value aromatic chemicals. The need to better characterize and understand this complex biopolymer has led to the development of many different analytical approaches, several of which involve mass spectrometry and subsequent data analysis. This review surveys the most important analytical methods for lignin involving mass spectrometry, first looking at methods involving gas chromatography, liquid chromatography and then continuing with more contemporary methods such as matrix assisted laser desorption ionization and time-of-flight-secondary ion mass spectrometry. Following that will be techniques that directly ionize lignin mixtures-without chromatographic separation-using softer atmospheric ionization techniques that leave the lignin oligomers intact. Finally, ultra-high resolution mass analyzers such as FT-ICR have enabled lignin analysis without major sample preparation and chromatography steps. Concurrent with an increase in the resolution of mass spectrometers, there have been a wealth of complementary data analyses and visualization methods that have allowed researchers to probe deeper into the "lignome" than ever before. These approaches extract trends such as compound series and even important analytical information about lignin substructures without performing lignin degradation either chemically or during MS analysis. These innovative methods are paving the way for a more comprehensive understanding of this important biopolymer, as we seek more sustainable solutions for our human species' energy and materials needs.

Keywords:  HRMS; analytical chemistry; biofuels; lignin; mass spectrometry

DOI:  https://doi.org/10.1002/mas.21716