Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

新型包覆Pd纳米粒子液相色谱-串联质谱法用于鉴定沙特阿拉伯原油中的含氧化合物(英文)

Saudi Arabian crude oil is a super complex mixture and,up to now,there has been little research into its heteroatom-containing compounds.First,oxygenated compounds(OCs)were isolated from Saudi Arabian oil using a Pd nanoparticle exchange complex,which formed between the nano-Pds and the oxygenated ligands.Normally,polycyclic aromatic sulphur heterocycles(S-PAHs)are separated from petroleum oil via the same method.The obtained results reveal that all the OC formulations with S-PAHs can be separated from the pre-isolated aromatic fraction of crude oil via this approach.S-PAHs are mixtures of benzothiophene and dibenzothiophene congeners.The isolated OCs are composed mainly of hydroxyl compounds.The liquid chromatography(LC)/electrospray ionization(ESI)in positive ion mode ESI(+)/tandem mass spectrometry(MS/MS)technique was used to assign the molecular weight distribution and identify the isolated OCs.The LC/ESI(+)-MS/MS technique differentiates S-PAHs and OCs using protonated ions.Thus,LC/ESI(+)-MS/MS can be used to assign molecular weight distributions for both the groups as a single mixture.MS/MS in precursor ion mode was used for the immediate identification of the target S or O analytes.     

新型包覆Pb纳米粒子液相色谱-串联质谱法用于鉴定沙特阿拉伯原油中的含氧化合物（英文）

Saudi Arabian crude oil is a super complex mixture and,up to now,there has been little research into its heteroatom-containing compounds.First,oxygenated compounds(OCs)were isolated from Saudi Arabian oil using a Pd nanoparticle exchange complex,which formed between the nano-Pds and the oxygenated ligands.Normally,polycyclic aromatic sulphur heterocycles(S-PAHs)are separated from petroleum oil via the same method.The obtained results reveal that all the OC formulations with S-PAHs can be separated from the pre-isolated aromatic fraction of crude oil via this approach.S-PAHs are mixtures of benzothiophene and dibenzothiophene congeners.The isolated OCs are composed mainly of hydroxyl compounds.The liquid chromatography(LC)/electrospray ionization(ESI)in positive ion mode ESI(+)/tandem mass spectrometry(MS/MS)technique was used to assign the molecular weight distribution and identify the isolated OCs.The LC/ESI(+)-MS/MS technique differentiates S-PAHs and OCs using protonated ions.Thus,LC/ESI(+)-MS/MS can be used to assign molecular weight distributions for both the groups as a single mixture.MS/MS in precursor ion mode was used for the immediate identification of the target S or O analytes.     

LC–DAD and LC–ESI–MS Chromatographic Fingerprinting and Quantitative Analysis for Evaluation of the Quality of Huang-Lian-Jie-Du-Tang

LC–DAD coupled with electrospray tandem mass spectrometry (LC–ESI–MS–MS) has been used to evaluate the quality of the traditional Chinese medicine Huang-Lian-Jie-Du-Tang (HLJDT). Twenty-five chromatographic peaks were obtained from a C₁₈ analytical column by gradient elution with acetonitrile and formate buffer (containing 0.5% formic acid) at a flow rate of 1.0 mL min^?1. The linearity, precision, and accuracy of the data obtained were acceptable. Thirteen components were identified by ESI–MS, and seven of these were quantitatively analyzed by LC–DAD. The method was used to analyze ten batches of HLJDT, and both chromatographic fingerprints and quantitative data were used to evaluate the quality of the HLJDT. It was concluded that this LC–DAD–ESI–MS method enables more fully validated and complete evaluation and monitoring of the quality of HLJDT.     

A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation

Reversed-phase microcapillary chromatography (RP-microLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) is one of two prevailing techniques in proteomic analysis, the other being matrix-assisted laser desorption/ionization (MALDI). Despite the arguably better dynamic range obtainable with ESI, MALDI is increasingly popular due to ease of use, ruggedness and the ability to decouple separation from ionization. By contrast, in order to take advantage of the sensitivity and dynamic range afforded by the concentration-dependent nature of ESI, it is directly coupled to separations that take place in small i.d. RP-microLC columns. This gain in sensitivity often comes at a loss of ruggedness due to clogging of the small i.d. RP-microLC columns, one result of which is limited sample throughput. Here we describe a combined micropre-column-microLC-ESI device that is sensitive, rugged and modular in design allowing facile construction and troubleshooting. Due to low signal-to-noise as little as 1 attomole of a peptide can be selected by data-dependent methods for collision-induced dissociation. Importantly, the resulting tandem mass spectrum is of high enough quality to identify the peptide sequence by a database search against a complex database using SEQUEST. Finally, the device is demonstrated to be rugged as judged by >60 consecutive reversed-phase microLC separations on complex peptide mixtures before chromatographic resolution is degraded.     

Ultrahigh-pressure dual online solid phase extraction/capillary reverse-phase liquid chromatography/tandem mass spectrometry (DO-SPE/cRPLC/MS/MS): a versatile separation platform for high-throughput and highly sensitive proteomic analyses

Capillary RPLC/ESI-MS (cRPLC/ESI-MS) is one of the most powerful analytical tools for current proteomic research. The development of cRPLC techniques coupled online to a mass spectrometer has focused on increasing the separation efficiency, detection sensitivity, and throughput. Recently, the use of high-pressure (over 10,000 psi) LC systems that utilize long, small inner diameter capillary columns has gained much attention for proteomic analyses. In this study, we developed an ultrahigh-pressure dual online SPE/capillary RPLC (DO-SPE/cRPLC) system. This LC system employs two online SPE columns and two capillary columns (75 microm inner diameter x 1 m length) in a single separation system, and has a maximum operating pressure of 10,000 psi. This DO-SPE/cRPLC system is capable of providing high-resolution separation in addition to several other advantageous features, such as high reproducibility in terms of the LC retention time, rapid sample injection, online desalting, online sample enrichment of dilute samples, and increased throughput as a result of essentially removing the column equilibration time between successive experiments. We coupled the DO-SPE/cRPLC system online to a tandem mass spectrometer to allow high-throughput proteomic analyses. In this paper, we demonstrate the efficiency of this DO-SPE/cRPLC/MS/MS system by its use in the analyses of proteomic samples exhibiting different levels of complexity.     

A validated LC–MS/MS method for fast detection of thiodiglycolic acid in aqueous samples

A novel sensitive screening method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has shown the feasibility of separation and detection of thiodiglycolic acid in aqueous samples. The analysis of this compound is of interest since it is specific microbiological metabolite of thiodiglycol, which is precursor and degradation product of chemical warfare agent sulfur mustard. The LC–electrospray ionisation (ESI)–MS method provides a sensitive and direct approach for thiodiglycolic acid identification and quantification using non-extracted non-derivitised samples from aqueous solutions. Chromatographic separation of the thiodiglycolic acid was produced using a reverse phase LC column with gradient mobile phases consisting of 0.1% formic acid in water and acetonitrile. Identification and quantification of species were achieved using ESI–tandem MS monitoring two precursor-to-product ion transitions for thiodiglycolic acid. The method demonstrates linearity over at least two orders of magnitude and detection limit of 10 ng...mL^–1 in environmental water samples.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

Ultra-performance liquid chromatography/tandem mass spectrometric quantification of structurally diverse drug mixtures using an ESI-APCI multimode ionization source

We report in this paper an ultra-performance liquid chromatography/tandem mass spectrometric (UPLC(R)/MS/MS) method utilizing an ESI-APCI multimode ionization source to quantify structurally diverse analytes. Eight commercial drugs were used as test compounds. Each LC injection was completed in 1 min using a UPLC system coupled with MS/MS multiple reaction monitoring (MRM) detection. Results from three separate sets of experiments are reported. In the first set of experiments, the eight test compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes (ESI+, ESI-, APCI-, and APCI+) during an LC run. Approximately 8-10 data points were collected across each LC peak. This was insufficient for a quantitative analysis. In the second set of experiments, four compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes during an LC run. Approximately 15 data points were obtained for each LC peak. Quantification results were obtained with a limit of detection (LOD) as low as 0.01 ng/mL. For the third set of experiments, the eight test compounds were analyzed as a batch. During each LC injection, a single compound was analyzed. The mass spectrometer was detecting at a particular ionization mode during each LC injection. More than 20 data points were obtained for each LC peak. Quantification results were also obtained. This single-compound analytical method was applied to a microsomal stability test. Compared with a typical HPLC method currently used for the microsomal stability test, the injection-to-injection cycle time was reduced to 1.5 min (UPLC method) from 3.5 min (HPLC method). The microsome stability results were comparable with those obtained by traditional HPLC/MS/MS.     

Targeted lipidomics using electron capture atmospheric pressure chemical ionization mass spectrometry

There is an increasing need to be able to conduct quantitative lipidomics analyses as a complement to proteomics studies. The highest specificity for proteomics analysis can be obtained using methodology based on electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS). For lipidomics analysis it is often necessary to be able to separate enantiomers and regioisomers. This can be very challenging when using methodology based on conventional reversed-phase chromatography. Normal-phase chromatography using chiral columns can provide dramatic improvements in the resolution of enantiomers and regioisomers. However, conventional ESI- and APCI-MS/MS has limited sensitivity, which makes it difficult to conduct studies in cell culture systems where only trace amounts of non-esterified bioactive lipids are present. The use of electron capture APCI-MS/MS overcomes this problem. Enantiomers and regioisomers of diverse bioactive lipids can be quantified using stable isotope dilution methodology coupled with normal-phase chiral chromatography and electron capture APCI-MS/MS. This methodology has allowed a lipidomics profile from rat epithelial cells maintained in culture to be delineated and allowed the effect of a non-selective lipoxygenase inhibitor to be assessed.     

Electrospray photoionization (ESPI) liquid chromatography/mass spectrometry for the simultaneous analysis of cyclodextrin and pharmaceuticals and their binding interactions

We report on the use of a multimode electrospray ionization/atmospheric pressure photoionization source (ESI/APPI or ESPI for short) with liquid chromatography/mass spectrometry (LC/MS) to measure all components of a mixed-polarity liquid sample containing: (1) low-polarity component (hormone, pharmaceutical or sterol), (2) polar component (cyclodextrin substrate), and (3) bound polar complex. The ESPI source has several advantages over both single ESI and multimode electrospray ionization/chemical ionization (ESCI) analysis, including an enhanced bound-complex detection and better performance at lower solvent flow rates. Relative binding constants are determined with (i) ESI mode, resulting in relative R(ESI-MS) values, and (ii) both ESI and APPI modes, providing relative K(D) values. We find that low molecular-substitution (Ms) values of cyclodextrin, i.e., Ms = 0.4, preferentially bind to the low-polarity compounds tested. This investigation is intended to demonstrate the feasibility of ESPI as an additional tool for investigating mixed-polarity binding systems, providing mass-specific data for all solution components, both polar and non-polar.     

High sensitivity determination of valproic acid in mouse plasma using semi-automated sample preparation and liquid chromatography with tandem mass spectrometric detection

A high-throughput liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) assay using automated sample preparation has been developed for the determination of valproic acid (VPA) in mouse plasma. A liquid-handling system was programmed to prepare calibration standard solutions in plasma, as well as quality controls and clinical samples. Plasma protein precipitation was performed on a 96-well plate, and the collected supernatant was directly injected into a reversed-phase LC/ESI-MS/MS system in the negative ionization mode. The calibration curve for VPA was linear over a dynamic range of 0.15-100 microg/mL. The limit of detection was 75 ng/mL and the lower limit of quantitation was 150 ng/mL. Intra- and inter-day validation assays of the semi-automated plasma analysis showed satisfactory accuracy and precision.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.     

Rapid screening and confirmation of drugs and toxic compounds in biological specimens using liquid chromatography/ion trap tandem mass spectrometry and automated library search

Recent advances in liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology have provided an opportunity for the development of more specific approaches to achieve the ‘screen’ and ‘confirmation’ goals in a single analytical step. For this purpose, this study adapts the electrospray ionization ion trap LC/MS/MS instrumentation (LC/ESI‐MS/MS) for the screening and confirmation of over 800 drugs and toxic compounds in biological specimens. Liquid‐liquid and solid‐phase extraction protocols were coupled to LC/ESI‐MS/MS using a 1.8‐µm particle size analytical column operated at 50°C. Gradient elution of the analytes was conducted using a solvent system composed of methanol and water containing 0.1% formic acid. Positive‐ion ESI‐MS/MS spectra and retention times for each of the 800 drugs and toxic compounds were first established using 1–10 µg/mL standard solutions. This spectra and retention time information was then transferred to the library and searched by the identification algorithm for the confirmation of compounds found in test specimens – based on retention time matches and scores of fit, reverse fit, and purity resulting from the searching process. The established method was found highly effective when applied to the analyses of postmortem specimens (blood, urine, and hair) and external proficiency test samples provided by the College of American Pathology (CAP). The development of this approach has significantly improved the efficiency of our routine laboratory operation that was based on a two‐step (immunoassay and GC/MS) approach in the past. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatography-electrospray ionization tandem mass spectrometry and liquid chromatography with fluorescence detection for determination of octylphenol and nonylphenol in municipal wastewater at trace levels

Summary Reversed-phase liquid chromatography with fluorescence detection (RPLC-FD) and with electrospray ionization (ESI) tandem mass spectrometric detection (RPLC-ESI-MS-MS), both coupled with automated solid-phase sample extraction, have been used for trace enrichment and analysis of octylphenol and nonylphenol (NP) in municipal wastewater. Quality data calculated for the methods were detection limits (LOD), quantitation limits, linearity, precision, and accuracy. For LC-ESI-MS-MS theLOD were below 0.08 μg L⁻¹. Unequivocal detection of NP in wastewater samples was achieved by use of LC-ESI-MS and LC-ESI-MS-MS. In particular, application of the LC methods to wastewater samples revealed that the selectivity of LC-MS-MS was more than that of LC-FD for qualitative and quantitative analysis of complex matrices.     

An alternative and fast method for determination of glyphosate and aminomethylphosphonic acid (AMPA) residues in soybean using liquid chromatography coupled with tandem mass spectrometry

A simple and specific method using reversed‐phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) was investigated, which allowed the determination of residues of glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), in soybean samples. An aqueous extraction with liquid‐liquid partition followed by protein precipitation was performed before the LC/MS/MS determination. The quantitation of glyphosate and AMPA was performed in positive and negative ESI mode, respectively, using the multiple reaction monitoring (MRM) mode with three transitions for each analyte to enhance the specificity of the method and avoid false positives. The methodology reported in this work is capable of detecting residues of glyphosate and AMPA in soybean samples with limits of quantification of 0.30 and 0.34 mg kg^?1, respectively. This alternative method has throughput advantages such as simpler sample preparation and faster chromatographic analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

A simultaneous determination method for 5‐fluorouracil and its metabolites in human plasma with linear range adjusted by in‐source collision‐induced dissociation using hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry

We applied a new technique for quantitative linear range shift using in‐source collision‐induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5‐fluorouracil (5‐FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS). To control adverse effects after administration of 5‐FU, it is important to monitor the plasma concentration of 5‐FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5‐FU and its metabolites in human plasma by LC/ESI‐MS/MS coupled with the technique for quantitative linear range shift using in‐source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5‐FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile‐rich eluent after LC separation improved the ESI‐MS response of high polar analytes. Based on the validation results, linear range shifts by in‐source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.     

Use of a fritless dual tapered column and a low flow interface for capillary electrochromatography-mass spectrometry

To avoid problems associated with the use of sintered frits to retain packing material, tapered columns were investigated for use with capillary electrochromatography-mass spectrometry (CEC-MS) analysis. Taking the advantage that negatively charged stationary phase particles have a net velocity directed towards the buffer reservoir (inlet) over a wide range in pH, a fritless CEC column with a single taper tip was prepared for CEC-MS analysis. During CEC-MS analysis, the tapered end was immersed in the buffer reservoir and the unmodified end was pointed toward the ionization source. For better sensitivity, this single tapered CEC column was coupled to ESI/MS using a low flow sheath liquid interface. With this setup, occasional blockage of the ESI sprayer by stationary phase particles was observed. In addition, significant dead volume was observed because the unmodified tip could not be inserted into the very end of the sprayer of the low flow sheath liquid interface. To circumvent these problems, a dual tapered CEC column was prepared. This fritless dual tapered column CEC-MS approach alleviated the problems of frit, sprayer blockage and extensive dead volume.     

An analytical strategy for identification of a somatotropin‐like bioactive peptide by ion trap liquid chromatography/electrospray ionization tandem mass spectrometry after immuno‐affinity purification from buffalo serum

The use of growth hormones, such as native and recombinant somatotropins, is forbidden in the European Union (EU), but is legal in the USA. The misuse of recombinant bovine somatotropin in Italy is suspected for enhancing milk production, thanks to its availability on the illegal market. A synthetic bioactive peptide of 27 amino acids derived from bovine somatotropin was successfully tested in France and in southern Italy for scientific purposes, to stimulate milk production, both in cows and buffaloes. This somatotropin‐like peptide (PEP‐ST), suspected for illegal use in southern Italy, was synthesized by linking the 104–113 sequence of bovine somatotropin to the 323–339 sequence of ovalbumin. Herein, a method for detection and identification of the PEP‐ST in buffalo serum is described; our strategy was based on the production of IgG anti‐PEP‐ST, used to synthesize an immuno‐affinity column for peptide purification from buffalo serum, prior to analysis by ion trap liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). The immuno‐affinity column was successfully used to purify in a single step the bioactive PEP‐ST from buffalo serum samples spiked at 20, 50 and 200 µg/mL for confirmatory analysis. Ion trap LC/ESI‐MS/MS identification was based on detection of a multi‐charged molecular ion and its characteristic fragmentation pattern. No significant matrix interference was observed, accounting for method specificity. We consider this strategy to be a basic approach that could be improved in the perspective of the official control of illegal use of somatotropin and somatotropin‐like compounds in buffalo breeding. Copyright © 2009 John Wiley & Sons, Ltd.