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Rationale: While natural products isolated from medicinal plants can serve as a rich source of biologically active metabolites, mixtures of structurally related compounds of a polar nature are often difficult to chemically resolve by traditional separation techniques. Chemical derivatization to reduce metabolite polarity combined with liquid chromatography (LC) is the strategy presented here to resolve a mixture of structurally related natural product glycosides solvent extracted from the medicinal herb Teucrium polium for mass spectrometric characterization., Methods: The partially purified plant extract was subjected to chemical derivatization and electrospray ionization mass spectrometry (ESI-MS) fragmentation pattern analysis allowed for structural characterization of iridoid and secoiridoid glycosides. Selected ions were subjected to tandem mass spectrometric (MS/MS) analysis with a relatively higher-energy collision dissociation to assist in structural elucidation., Results: Permethylation replaced all protons from free hydroxyl and amino groups with methyls and resulted in increases in both hydrophobicity, for facilitated chromatographic separation, and proton affinity, for enhanced chemical ionization. Protonated and/or sodiated adducts were observed for the six compounds detected in positive-ion mode ESI-MS with a mass accuracy of less than 2 ppm., Conclusions: Permethylation combined with LC/MS analysis is shown here to be an effective chemical practice for separating and characterizing iridoid glucosinolates and is expected to be well suited for the chemical characterization of other polar natural-product mixtures of closely related compounds. Copyright © 2016 John Wiley & Sons, Ltd., Competing Interests: The authors declare no conflict of interest., (Copyright © 2016 John Wiley & Sons, Ltd.)

Rationale: Liquid chromatography/mass spectrometry (LC/MS) is currently considered to be a conventional glycomics analysis strategy due to the high sensitivity and ability to handle complex biological samples. Interpretation of LC/MS data is a major bottleneck in high-throughput glycomics LC/MS-based analysis. The complexity of LC/MS data associated with biological samples prompts the needs to develop computational tools capable of facilitating automated data annotation and quantitation., Methods: An LC/MS-based automated data annotation and quantitation software, MultiGlycan-ESI, was developed and utilized for glycan quantitation. Data generated by the software from LC/MS analysis of permethylated N-glycans derived from fetuin were initially validated by manual integration to assess the performance of the software. The performance of MultiGlycan-ESI was then assessed for the quantitation of permethylated fetuin N-glycans analyzed at different concentrations or spiked with permethylated N-glycans derived from human blood serum., Results: The relative abundance differences between data generated by the software and those generated by manual integration were less than 5%, indicating the reliability of MultiGlycan-ESI in quantitation of permethylated glycans analyzed by LC/MS. Automated quantitation resulted in a linear relationship for all six N-glycans derived from 50 ng to 400 ng fetuin with correlation coefficients (R(2) ) greater than 0.93. Spiking of permethylated fetuin N-glycans at different concentrations in permethylated N-glycan samples derived from a 0.02 μL of HBS also exhibited linear agreement with R(2) values greater than 0.9., Conclusions: With a variety of options, including mass accuracy, merged adducts, and filtering criteria, MultiGlycan-ESI allows automated annotation and quantitation of LC/ESI-MS N-glycan data. The software allows the reliable quantitation of glycan LC/MS data. The software is reliable for automated glycan quantitation, thus facilitating rapid and reliable high-throughput glycomics studies., (Copyright © 2014 John Wiley & Sons, Ltd.)

Rationale: Glycosylation of proteins and lipids is reliant on the availability of monosaccharide-activated donors known as sugar nucleotides. They are responsible for glycosylation in cells. Reliable quantification of these sugar nucleotides might provide an insight into their biological roles and attributes., Methods: Herein, a method is described for the quantification of sugar nucleotides using ultra high pressure liquid chromatography (UHPLC) tandem mass spectrometry, allowing selective detection of sugar nucleotides in a biological sample. Seven model sugar nucleotide standards commonly associated with lipid and protein glycosylation were separated on a porous graphitic carbon column using an UHPLC system coupled to a triple stage quadrupole mass spectrometer utilizing a multiple reaction monitoring approach., Results: Successful baseline separation of these metabolites was attained in 6 min using an ammonium formate buffer and acetonitrile, circumventing the use of MS-unfriendly pairing reagents. The linear dynamic range of this procedure was established over almost three orders of magnitude from 20 pg to 1 ng (40 pg to 2 ng for the isomers UDP-GlcNAc/GalNAc). The limit of detection ranged from 15 pg to 30 pg while the limit of quantification ranged from 50 pg to 100 pg. Furthermore, viability of this method was tested using three different breast cancer cell lines (MDA-MB-231, MDA-MB-231-BR, and MDA-MB-361) with the successful identification and quantification of all seven targeted sugar nucleotides., Conclusions: The described method permitted the quantitative analysis of sugar nucleotides in 10 min, thus allowing the practical use of this approach in high-throughput settings. The method was also very effective for the quantification of sugar nucleotides derived from three different breast cancer cell lines. The distribution of sugar nucleotides was different among the different cell lines and unique for each cell line., (Copyright © 2013 John Wiley & Sons, Ltd.)

Rationale: Mass spectrometry based comparative glycomics is essential for disease biomarker discovery. However, developing a reliable quantification method is still a challenging task., Methods: We here report an isotopic labeling strategy employing stable isotopic iodomethane for comparative glycomic profiling by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). N-Glycans released from model glycoproteins and blood serum samples were permethylated with iodomethane ('light') and iodomethane-d1 or -d3 ('heavy') reagents. Permethylated samples were then mixed at equal volumes prior to LC/ESI-MS analysis., Results: Peak intensity ratios of N-glycans isotopically permethylated (Heavy/Light, H/L) were almost equal to the theoretical values. Observed differences were mainly related to the purity of 'heavy' iodomethane reagents (iodomethane-d1 or -d3). The data suggested the efficacy of this strategy to simultaneously quantify N-glycans derived from biological samples representing different cohorts. Accordingly, this strategy is effective in comparing multiple samples in a single LC/ESI-MS analysis. The potential of this strategy for defining glycomic differences in blood serum samples representing different esophageal diseases was explored., Conclusions: LC/ESI-MS comparative glycomic profiling of isotopically permethylated N-glycans derived from biological samples and glycoproteins reliably defined glycan changes associated with biological conditions or glycoproteins expression. As a biological application, this strategy permitted the reliable quantification of glycomic changes associated with different esophageal diseases, including high grade dysplasia, Barrett's disease, and esophageal adenocarcinoma., (Copyright © 2013 John Wiley & Sons, Ltd.)

Protein glycosylation has a major influence on functions of proteins. Studies have shown that aberrations in glycosylation are indicative of disease conditions. This has prompted major research activities for comparative studies of glycoproteins in biological samples. Multiple reaction monitoring (MRM) is a highly sensitive technique which has been recently explored for quantitative proteomics. In this work, MRM was adopted for quantification of glycopeptides derived from both model glycoproteins and depleted human blood serum using glycan oxonium ions as transitions. The utilization of oxonium ions aids in identifying the different types of glycans bound to peptide backbones. MRM experiments were optimized by evaluating different parameters that have a major influence on quantification of glycopeptides, which include MRM time segments, number of transitions, and normalized collision energies. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, the specificity increased with the number of transitions and a more sensitive analysis can be obtained by providing specific time segments. This approach can be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum sample., (Copyright © 2012 John Wiley & Sons, Ltd.)

Protein glycosylation is one of the most common post-translational modifications, estimated to occur in over 50% of human proteins. Mass spectrometry (MS)-based approaches involving different fragmentation mechanisms have been frequently used to detect and characterize protein N-linked glycosylations. In addition to the popular Collision-Induced Dissociation (CID), high-energy C-trap dissociation (HCD) fragmentation, which is a feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap), has been recently used for the fragmentation of tryptic N-linked glycopeptides in glycoprotein analysis. The oxonium ions observed with high mass accuracy in the HCD spectrum of glycopeptides can be combined with characteristic fragmentation patterns in the CID spectrum resulting from consecutive glycosidic bond cleavages, to improve the detection and characterization of N-linked glycopeptides. As a means of automating this process, we describe here GlypID 2.0, a software tool that implements several algorithmic approaches to utilize MS information including accurate precursor mass and spectral patterns from both HCD and CID spectra, thus allowing for an unequivocal and accurate characterization of N-linked glycosylation sites of proteins., (Copyright © 2011 John Wiley & Sons, Ltd.)

Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones., (Copyright © 2010 John Wiley & Sons, Ltd.)

A simple, sensitive, and reproducible quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was designed for the simultaneous quantification of monosaccharides derived from glycoprotein and blood serum using a multiple-reaction monitoring (MRM) approach. Sialic acids and neutral monosaccharides were efficiently separated using an amino-bonded silica phase column. Neutral monosaccharide molecules were detected as their aldol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids were detected as deprotonated ions [M-H](-). The new method did not require a reduction step, and exhibited very high sensitivity to carbohydrates with limits of detection of 1 pg for the sugars studied. The linearity of the described approach spanned over three orders of magnitude (pg to ng). The method was validated for monosaccharides originating from N-linked glycans attached to glycoproteins and glycoproteins found in human blood serum. The method effectively quantified monosaccharides originating from as little as 1 microg of glycoprotein and 5 microL of blood serum. The method was robust, reproducible, and highly sensitive. It did not require reduction, derivatization or postcolumn addition of reagents., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

Assigning glycosylation sites of glycoproteins and their microheterogeneity is still a very challenging analytical task despite the rapid advancements in mass spectrometry. It is shown here that glycopeptide ions can be fragmented efficiently using the higher-energy C-trap dissociation (HCD) feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap). An attractive aspect of this dissociation option is the generation of distinct Y1 ions (peptide+GlcNAc), thus allowing unequivocal assignment of N-glycosylation sites of glycoproteins. The combination of the very informative collision-induced dissociation spectra acquired in the linear ion trap with the distinct features of HCD offers very useful information aiding in the characterization of the glycosylation sites of glycoproteins. The HCD activation energy needed to obtain optimum Y1 ions was studied in terms of glycan structure and charge state, and size and structure of the peptide backbone. The latter appeared to be primarily dictating the needed HCD energy. The distinct Y1 ion formation in HCD facilitated an easy assignment of such an ion and its subsequent isolation and dissociation through multiple-stage tandem mass spectrometry. The resulting MS(3) spectrum of the Y1 ion facilitates database searching and de novo sequencing thus prompting the subsequent identification of the peptide backbone and associated glycosylation sites. Moreover, fragment ions formed by HCD are detected in the Orbitrap, thus overcoming the 1/3 cut-off limitation that is commonly associated with ion trap mass spectrometers. As a result, in addition to the Y1 ion, the common glycan oxonium ions are also detected. The high mass accuracy offered by the LTQ Orbitrap mass spectrometer is also an attractive feature that allows a confident assignment of protein glycosylation sites and the microheterogeneity of such sites., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

Glycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestion followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, it is often not possible to characterize all glycopeptides in a complex sample because of the high complexity of glycoproteomic samples, and the relative lower abundances of glycopeptides in comparison to the unmodified peptides. We present here a targeted MS/MS analysis approach, which utilizes a previously developed computational tool, GlyPID, to guide multiple experiments, thus permitting a complete characterization of all N-glycosylation sites of glycoproteins present in a complex sample. We have tested our approach using model glycoproteins analyzed by high-resolution LTQ-FT MS. The results demonstrate a potential use of our method for a high-throughput characterization of complex mixtures of glycosylated proteins., (2010 John Wiley & Sons, Ltd.)

The difference in serum phospholipid content between stage-IV breast cancer patients and disease-free individuals was studied by employing a combination of chemometric statistical analysis tools and mass spectrometry. Chloroform-extracted serum samples were profiled for their lipid class composition and structure using precursor ion, neutral loss, and product ion tandem mass spectrometric (MS/MS) scanning experiments. Changes in the relative abundance of phospholipids in serum as a consequence of cancer progression, measured through electrospray ionization (ESI) mass spectrometry of flow-injected serum samples collected from 25 disease-free individuals and 50 patients diagnosed with stage-IV breast cancer, were statistically evaluated using principal component analysis (PCA), analysis of variance (ANOVA) and receiver operating characteristic (ROC) analysis. Lipids whose abundance changed significantly as a consequence of cancer progression were structurally characterized using product ion spectra, and independently quantified using precursor ion scan experiments against an internal standard of known concentration. Phosphocholine lipids that displayed a statistically significant change as a consequence of cancer progression were found to contain an oxidized fatty acid moiety as determined by MS3 experiments., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip-based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C(18) LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C(18) chips. On the other hand, hydrophobic glycopeptides were better retained on C(18) chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1 pmol). The performance of both media also appeared comparable when analyzing a four-protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase., (Copyright 2009 John Wiley & Sons, Ltd.)

Structural characterization of a glycopeptide is not easily attained through collision-induced dissociation (CID), due to the extensive fragmentation of glycan moieties and minimal fragmentation of peptide backbones. In this study, we have exploited the potential of electron-transfer dissociation (ETD) as a complementary approach for peptide fragmentation. Model glycoproteins, including ribonuclease B, fetuin, horseradish peroxidase, and haptoglobin, were used here. In ETD, radical anions transfer an electron to the peptide backbone and induce cleavage of the N-Calpha bond. The glycan moiety is retained on the peptide backbone, being largely unaffected by the ETD process. Accordingly, ETD allows not only the identification of the amino acid sequence of a glycopeptide, but also the unambiguous assignment of its glycosylation site. When data acquired from both fragmentation techniques are combined, it is possible to characterize comprehensively the entire glycopeptide. This is being achieved with a mass spectrometer capable of alternating between CID and ETD on-the-fly during an LC/MS/MS analysis. This is demonstrated here with several tryptic glycopeptides., ((c) 2008 John Wiley & Sons, Ltd.)

In simplifying the evaluation and quantification of high-throughput label-free quantitative proteomic data, we introduce ProteinQuant Suite. It comprises three standalone complementary computer utilities, namely ProtParser, ProteinQuant, and Turbo RAW2MGF. ProtParser is a filtering utility designed to evaluate database search results. Filtering is performed according to different criteria that are defined by the end-user. ProteinQuant then utilizes this parsed list of peptides and proteins in conjunction with mzXML or mzData files generated from the raw files for quantification. This quantification is based on the automatic detection and integration of chromatographic peaks representative of the liquid chromatography/mass spectrometry (LC/MS) elution profiles of identified peptides. Turbo RAW2MGF was developed to extend the applicability of ProteinQuant Suite to data collected from different types of mass spectrometers. It directly processes raw data files generated by Xcalibur, a ThermoElectron data acquisition software, and generates a MASCOT generic file (MGF). This file format is needed since the protein identification results generated by the database search employing this file format include information required for the precise identification and quantification of chromatographic peaks. The performance of ProteinQuant Suite was initially validated using LC/MS/MS generated for a mixture of standard proteins as well as standard proteins spiked in a complex biological matrix such as blood serum. Automated quantification of the collected data resulted in calibration curves with R(2) values higher than 0.95 with linearity spanning over more than 2 orders of magnitude with peak quantification reproducibility better than 15% (RSD). ProteinQuant Suite was also applied to confirm the binding preference of standard glycoproteins to Con A lectin using a sample consisting of both standard glycoproteins and proteins.

Glycans commonly exhibit variations in their branching structures and glycosidic bond linkages, in addition to their sequence variation. These glycan features are known to be highly correlated with their biological functions. It is relatively straightforward to deduce the composition and sequence of monosaccharides of a glycan from its tandem mass spectra. However, the characterization of the linkage types of each glycosidic bond is still analytically challenging. In this paper, we present a rank-based discriminative model to differentiate between two types of glycosidic linkages (namely, 1-4 and 1-6) based on the cross-ring fragmentation patterns of the corresponding glycans observed under high-energy collision-induced dissociation (CID). To train our models, we acquired tandem mass spectra for three groups of both native and permethylated linear oligoglucoses using a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI/TOF/TOF) instrument. Based on a 5-fold cross-validation, the prediction accuracies of our model for native glycans are determined to be about 88.4% and 92.9% for 1-6 and 1-4 linkages, respectively. The accuracies determined for permethylated glycans are slightly lower, but comparable: 85.6% and 89.0% for 1-6 and 1-4 linkages, respectively. Our method is implemented as a web-hosted utility, thus making it readily accessible to the public which can be accessed through http://ggdb.informatics.indiana.edu:8080/glycanview.

Permethylation of glycans prior to their mass spectrometric determination has now become a time-honored methodology in glycoconjugate analysis due to the advantage of a simultaneous analysis of neutral and acidic glycans as well as enhanced sensitivity and easier tandem mass spectrometry interpretation. While the different solvent extraction-based versions of this method often suffice in different structural studies, they are generally less satisfactory in the quantitative determinations aiming at minor quantities of the analyzed materials. To overcome these difficulties, we recently introduced a solid-phase capillary permethylation technique (Kang et al., Rapid Commun. Mass Spectrom. 2005; 19: 3421) for microscale determination. Here, we describe a very useful high-throughput extension of the solid-phase methodology utilizing spin columns packed with sodium hydroxide beads. This procedure has been thoroughly optimized to match the analytical performance parameters of the previously used capillary technique. As demonstrated with a high-precision glycomic profiling analysis of human blood serum, this methodological improvement offers simplicity and reproducibility, allowing the complete permethylation of 12-18 samples in less than 20 min.

Permethylated acidic and neutral N-glycans representing different types of glycan structures, such as linear and branched sialylated structures, high-mannose type and fucosylated complex type, were photodissociated with 157 nm vacuum ultraviolet light in a linear ion trap. Cross-ring fragments corresponding to high-energy fragmentation pathways were observed in abundance for all studied structures. Some product ions appear diagnostic for a linkage of sialic acid residues and the glycan antenna to which these residues are attached. A conclusive assignment of the fucosylation site of the studied glycan structure has been facilitated through measurement of cross-ring fragmentation resulting from photodissociation., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Using model acidic glycans, we demonstrate the benefits of permethylation for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) tandem mass spectrometry. With both the linear and branched structures, extensive cross-ring fragmentation product ions were generated, yielding valuable information on sugar linkages. Elimination of the negative charges commonly associated with sialylated structures through permethylation allowed their structural analysis in the positive ion mode. Extensive A- and X-type ions were observed for the linear structures, and slightly weaker signals for the branched sialylated structures. The diagnostic cross-ring fragments, permitting a distinction between alpha2-3 and alpha2-6 linkages of the sialic acid residues, were seen in abundance. Importantly, the cross-ring fragmentation with the branched structures provides adequate information to assign sialic acid residues, with a specific linkage, to a particular antenna., (Copyright 2006 John Wiley & Sons, Ltd.)

A miniaturized approach was developed for quantitative permethylation of oligosaccharides, which involves packing of sodium hydroxide powder in microspin columns or fused-silica capillaries (500 microm i.d.), permitting effective derivatization in less than a minute at microscale. Prior to mass spectrometry, analytes are mixed with methyl iodide in dimethyl sulfoxide solution containing traces of water before infusing through the microreactors. This procedure minimizes oxidative degradation and peeling reactions and avoids the need of excessive clean-up. Picomole amounts of linear and branched, sialylated and neutral glycan samples were rapidly and efficiently permethylated by this approach and analyzed by matrix-assisted laser desorption/ionization mass spectrometry.

Post-source decay (PSD) fragmentation of chemically or enzymatically produced aminoglycans has been evaluated through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Conversion of native glycans to their respective aminoglycan derivatives improved detection sensitivity of the usual fragments and promoted cross-ring fragmentation of linear oligosaccharides, facilitating linkage recognition. The cross-ring fragmentations for both dextrin and dextran oligosaccharides were not limited to the reducing-end glucose moiety, as they were extended throughout the entire molecule. When the amino group was generated for N-glycans derived from three different glycoproteins, an enhancement of PSD was observed, without a significant extent of cross-ring fragmentation., (Copyright 2004 John Wiley & Sons, Ltd.)

A new beta-elimination procedure has been introduced to cleave O-linked oligosaccharides from low- to sub-microgram amounts of glycoproteins prior to analysis by mass spectrometry. Borane-ammonia complex in aqueous ammonia is used as a cleaving solution alternative to the sodium borohydride/sodium hydroxide medium conventionally used in beta-elimination. The procedure results in minimum sample purification, leading to minimal sample loss and consequently an overall enhancement in sensitivity. It was applied successfully in the analysis of bovine fetuin and submaxillary mucin, as well as to a complex bile-salt-stimulated lipase glycoprotein isolated from human milk., (Copyright 2002 John Wiley & Sons, Ltd.)

A simple microadsorption technique is described to remove detergent additives from oligosaccharide samples before their mass spectrometric analysis. The described methodology has been validated with submicrogram quantities of contaminated glycoproteins. This procedure is applicable to investigating minute quantities of glycans in both the positive- and negative-ion mode of matrix-assisted laser desorption/ionization mass spectrometry., (Copyright 2000 John Wiley & Sons, Ltd.)