Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.

Factors associated with Giardia infection in dogs in southern Ontario, Canada.

Information concerning risk factors associated with Giardia infection in dogs in southern Ontario, Canada, is currently lacking. This study therefore aimed to identify risk factors for Giardia infection in dogs that visit off-leash dog parks in southern Ontario. From May-November 2018, fecal samples were collected from 466 dogs in 12 off-leash dog parks in the Niagara and Hamilton regions of Ontario. A survey that asked questions pertaining to travel history (i.e., area of residence, locations and regions visited in the previous 6 months), basic medical history (i.e., spay/neuter status, veterinary visits, use of deworming medication), consumption of a raw diet, and the physical (i.e., age, sex, breed) and behavioral characteristics (i.e., off-leash activities, hunting activities) of each dog sampled was administered to the respective owner. All fecal samples were examined with the Giardia plate ELISA (IDEXX Laboratories) for parasite antigen. Multivariable logistic regression analyses were conducted on the survey data to investigate putative risk factors for Giardia infection. Overall, 11.8% (95% CI: 9.2-15.1%) of samples tested positive for Giardia antigen. Results from the multivariable logistic regression analyses identified an interaction between dog age and spay/neuter status that was significantly associated with Giardia infection. The odds of infection were greater in intact as compared to neutered adult dogs (OR: 3.6, 95% CI: 1.7-7.9, p = 0.001), and in neutered juvenile dogs as compared to neutered adults (OR: 5.2, 95% CI: 2.2-12.2, p < 0.001). The results provide veterinarians with evidence-based information for identifying dogs at greatest risk of Giardia infection in southern Ontario.

Maximizing hydrophobic peptide recovery in proteomics and antibody development using a mass spectrometry compatible surfactant.

Peptide loss due to surface absorption can happen at any step in a protein analysis workflow and is sometimes especially deleterious for hydrophobic peptides. In this study, we found the LC-MS compatible surfactant, n-Dodecyl-ß-D-maltoside (DDM), can maximize hydrophobic peptide recovery in various samples including single cell digests, mAb clinical PK samples, and mAb peptide mapping samples. In HeLa single cell proteomics analysis, more than half of all unique peptides identified were found only in DDM prepared samples, most of which had significantly higher hydrophobicities compared to peptides in control samples. In clinical PK studies, DDM enhanced hydrophobic complementarity-determining region (CDR) peptide signals significantly. The fold change of CDR peptides' intensity enhancement in DDM added samples compared to controls correlate with peptide retention time and hydrophobicity, providing guidance for surrogate peptide selection and peptide standard handling in PK studies. For peptide mapping analysis of mAbs, DDM can improve hydrophobic peptide signal and solution stability over 48 h in an autosampler at 4 °C, which can aid method qualification and transfer during drug development. Lastly, maximizing hydrophobic peptide recovery from samples dried in vacuo was achieved by DDM reconstitution, which provided higher signal for later eluting peaks and higher proteome coverage overall.

High throughput and high confidence sequence variant analysis in therapeutic antibodies using Evosep One liquid chromatography tandem mass spectrometry with synthetic heavy peptides.

Sequence variants are anomalous misincorporations of amino acids into the primary structure of therapeutic antibodies during DNA replication and protein biosynthesis. As these low abundance variants contribute to molecular heterogeneity and could negatively impact the safety and efficacy of a protein therapeutic, analytical methods like liquid chromatography tandem mass spectrometry (LC-MS2) are used to monitor them with the goal of establishing control strategies that limit their occurrence. Current LC-MS2 strategies depend on relatively long gradients that minimize coelution between abundant non-variant peptide peaks and trace-level variants to limit ion suppression that can potentially conceal the latter. However, lengthy LC gradients reduce the number of samples that can be analyzed per day, limiting the practicality of LC-MS2 when analyzing large sample sets. Furthermore, confident variant identification partly depends on capturing rich MS2 spectra that localize any amino acid misincorporations, which can be challenging due to the low abundance of this class of analyte. This work drastically reduces the cycle time to run each therapeutic antibody sample with roughly the same or even more variant identifications, compared to traditional LC-MS2 analysis, by integrating an Evosep One LC platform with an Orbitrap Fusion Lumos mass spectrometer. It also introduces a novel strategy using synthetic peptides that contain heavy isotopes placed near both termini to validate lower confidence variants in one targeted LC-MS2 run according to retention time, precursor mass signal, and MS2 fragment patterns shared with the heavy peptide variant. Taken together, this approach enables high-throughput sequence variant analysis at 30 samples per day as well as validation for lower confidence variants that can be integrated into therapeutic antibody process development and characterization.

GIANT RED KIDNEY WORM (DIOCTOPHYMA RENALE) INFECTION IN PUPPIES LESS THAN FOUR MONTHS OF AGE FROM NORTHERN CANADA.

In this article, we present 2 case reports of puppies less than 4 mo of age at the time of infection with the nematode known as giant red kidney worm (Diotophyma renale). The first puppy was prepared for an ovariohysterectomy that evolved into an exploratory laparotomy. An inflamed omentum was observed, and D. renale infection was suspected due to the puppies' place of origin. The algorithm developed by the Ontario Society for the Prevention of Cruelty to Animals for the diagnosis and treatment of D. renale infections in dogs was followed. Both puppies had a history of origin from northern Manitoba, Canada. Due to the puppies' ages, the authors suspect that the transmission of infection for both puppies could be transmammary, transplacental, or ingestion of paratenic hosts or that these cases challenge what is thought to be the current prepatent period. In addition, D. renale infections in dogs, especially from northern communities, could be common.

Development of a simple non-reduced peptide mapping method that prevents disulfide scrambling of mAbs without affecting tryptic enzyme activity.

Non-reduced peptide mapping by liquid chromatography-mass spectrometry (LC-MS) analysis is a commonly used method for disulfide linkage characterization to assess structural integrity and quality of therapeutic monoclonal antibodies (mAbs). However, disulfide scrambling artifacts induced during sample preparation are often observed when basic pH and high temperatures are used during denaturation and digestion. To minimize disulfide scrambling artifacts, methods using various acidic pH conditions have been developed by multiple groups. However, lower pH conditions increase missed and non-specific cleavages, which complicates disulfide bond analysis because the majority of enzymes used in protein characterization are most efficient at alkaline pH. Here, we developed a non-reduced peptide mapping method for mAb characterization that minimizes disulfide scrambling at basic pH by adding an oxidizing agent, cystamine, and a low concentration of iodoacetamide (IAA) alkylating agent. Two human IgG1 mAbs, one with kappa light chain and another one with lambda light chain, were used as model proteins to develop and optimize the method. Using this novel method, disulfide scrambled peptides related to light chain-heavy chain (LC-HC) inter-disulfide disruption were significantly reduced with high reproducibility compared to conventional methods. Results demonstrated that the cystamine-added method is robust and minimizes disulfide scrambling artifacts produced during sample preparation.

Prevalence of intestinal parasites in dogs in southern Ontario, Canada, based on fecal samples tested using sucrose double centrifugation and Fecal Dx® tests.

In southern Ontario, Canada, there is a lack of information concerning the prevalence of intestinal parasites in dogs. As such, this study aimed to characterize the prevalence of intestinal parasites in dogs visiting off-leash parks in the region using sucrose double centrifugation and Fecal Dx® tests. Additionally, data obtained via the sucrose double centrifugation method were used to evaluate the performance of the Fecal Dx® tests. Fecal samples were collected from 466 dogs aged ≥6 months from May to November 2018 (mean age = 3.7 years). Overall, eleven intestinal parasites were identified using sucrose double centrifugation. Roundworm eggs (Toxocara canis and Baylisascaris procyonis), hookworm eggs (Ancylostoma caninum and Uncinaria stenocephala), and whipworm eggs (Trichuris vulpis) were identified in 1.07% (95% confidence interval [CI] 0.38-2.56%), 5.79% (95% CI 3.85-8.31%), and 5.15% (95% CI 3.33-7.57) of samples, respectively. Using the Fecal Dx® tests, 1.07% (95% CI 0.38-2.56%), 4.29% (95% CI 2.64-6.55%), and 2.15% (95% CI 1.03-3.91) of the samples tested positive for roundworm, hookworm, and whipworm antigen, respectively. To assess the level of agreement between the Fecal Dx® tests and sucrose double centrifugation, three methods were used. Cohen's kappa indicated a fair-to-moderate level of agreement between Fecal Dx® tests and sucrose double centrifugation. In contrast, the prevalence-adjusted bias-adjusted kappa and Gwet's first-order agreement coefficient indicated almost perfect agreement between these tests, ranging from 0.87 to 0.99 among the parasites examined. This study provides valuable information on the prevalence of intestinal parasites in mature dogs in southern Ontario that will help guide parasite control recommendations for dogs in this region.

Giant Red Kidney Worm (Dioctophyma renale) Screening and Treatment Protocol and Aberrant Worm Migration In Dogs From Ontario and Manitoba, Canada.

The life cycle of Dioctophyma renale involves an intermediate host (oligochaete), a paratenic hosts (fish and frogs), and a definitive host (mustelids and canids). Dogs are at risk of infection with D. renale when they consume paratenic hosts infected with the larval form of D. renale. Water containing the oligochaete intermediate host cannot be disregarded as another source of infection. Infections occur mainly in the right kidney, but worms have also been found in the abdominal cavity as well as other organs. Most dogs appear asymptomatic and infections are usually noted as incidental findings on necropsy. Recently, the Ontario Society for the Prevention of Cruelty to Animals (SPCA) and Humane Society conducted transports of dogs located in northern remote communities. In 2016, some female dogs were found to be infected with D. renale upon ovariohysterectomy. In response to this discovery, we developed a screening protocol to screen for D. renale infections. In 2018, a total of 130 intact dogs were transferred from 2 northern communities in the provinces of Ontario and Manitoba. A prevalence of 7.94% (95% confidence interval 3.87-14.11%) was found from dogs from the northern communities. The screening protocol we developed provides a method of screening for dogs that are transported from communities that could be at risk of infection with D. renale.

Simple and Sensitive Method for Deep Profiling of Host Cell Proteins in Therapeutic Antibodies by Combining Ultra-Low Trypsin Concentration Digestion, Long Chromatographic Gradients, and BoxCar Mass Spectrometry Acquisition.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for the analysis of host cell proteins (HCP) during antibody drug process development due to its sensitivity, selectivity, and adaptability. However, the enormous dynamic range between the therapeutic antibody and accompanying HCPs poses a significant challenge for LC-MS based detection of these low abundance impurities. To address this challenge, enrichment of HCPs via immunoaffinity, protein A, 2D-LC, or other strategies is typically performed. However, these enrichments are time-consuming and sometimes require a large quantity of sample. Here, we report a simple and sensitive strategy to analyze HCPs in therapeutic antibody samples without cumbersome enrichment by combining an ultra-low trypsin concentration during digestion under nondenaturing conditions, a long chromatographic gradient, and BoxCar acquisition (ULTLB) on a quadrupole-Orbitrap mass spectrometer. Application of this strategy to the NIST monoclonal antibody standard (NISTmAb) resulted in the identification of 453 mouse HCPs, which is a significant increase in the number of identified HCPs without enrichment compared to previous reports. Known amounts of HCPs were spiked into the purified antibody drug substance, demonstrating that the method sensitivity is as low as 0.5 ppm. Thus, the ULTLB method represents a sensitive and simple platform for deep profiling of HCPs in antibodies.

Integration of liquid chromatography mass spectrometry with a heavy peptide response curve accurately measures unprocessed C-terminal lysine during peptide mapping analysis of therapeutic antibodies in a single run.

Therapeutic monoclonal and bispecific antibodies are susceptible to modification after protein biosynthesis. These post-translational modifications (PTMs) not only contribute to mass and charge heterogeneity, but they can also negatively impact the molecule's activity, half-life, and immunogenicity. Therefore, identification and quantification of PTMs are critical to ensure the safety and efficacy of an antibody therapeutic as well as demonstrate product consistency and process control. Unprocessed C-terminal lysine on the heavy chain (HC) is a prevalent modification that contributes to this charge heterogeneity in antibodies. Peptide mapping through liquid chromatography tandem mass spectrometry (LC-MS2) enjoys higher selectivity and sensitivity for measuring this PTM relative to global PTM methods, but differences in the ionization efficiencies of the unprocessed C-terminal K peptide and the truncated C-terminal K peptide result in its overestimation. Consequently, large discrepancies in this PTM's measured abundance may exist between different characterization assays used in regulatory filings, which can be further compounded by large variability when multiple mass spectrometers are used to quantify C-terminal K during a therapeutic's lifespan. In this study, we propose a simple new method to quantify unprocessed C-terminal K in antibodies in a single LC-MS2 run that incorporates heavy isotopic standards for both the unprocessed and truncated C-terminal K peptide to build a response curve and correct for the disparity in ionization efficiency between these two different peptide sequences. The approach was evaluated across two different Orbitrap-based mass spectrometers using multiple monoclonal and bispecific therapeutic antibodies, resulting in accurate (<10% error, as determined with peptide standards) and precise C-terminal K quantification during peptide mapping analysis.

Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of Monoclonal Antibodies.

Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry (MS2) can provide detailed insight into their primary structure with minimal sample preparation. The middle-down approach uses an enzyme to cleave mAbs into Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid chromatography tandem mass spectrometry (RPLC-MS2). As maximum sequence coverage is desired to obtain meaningful structural information at the subunit level, a host of dissociation methods have been developed, and sometimes combined, to bolster fragmentation and increase the number of identified fragments. Here, we present a design of experiments (DOE) approach to optimize MS2 parameters, in particular those that may influence electron transfer dissociation (ETD) efficiency to increase the sequence coverage of antibody subunits. Applying this approach to the NIST monoclonal antibody standard (NISTmAb) using three RPLC-MS2 runs resulted in high sequence coverages of 67%, 67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply this DOE strategy to model the parameters required to maximize the number of fragments produced in "low", "medium", and "high" mass ranges, which ultimately resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high sequence coverage percentages utilizing only one fragmentation method, ETD, and could be extended to other state-of-the-art techniques that combine multiple fragmentation mechanisms to increase coverage.

Evaluation of the prevalence of Echinococcus multilocularis in dogs that visit off-leash dog parks in southern Ontario, Canada.

Prior to 2012, Echinococcus multilocularis was not known to occur in any host in Ontario, Canada. However, since that year, five cases of alveolar echinococcosis have been diagnosed in dogs that resided at the western end of Lake Ontario. In addition, E. multilocularis has been shown to be a common infection in wild canids (i.e. coyotes and foxes) across southern Ontario with a high-risk infection cluster in the area surrounding the western shores of Lake Ontario and northern shores of Lake Erie. In regions endemic for E. multilocularis, dog ownership is considered a risk factor for human alveolar echinococcosis. A study was therefore carried out to determine the prevalence of E. multilocularis intestinal infections in dogs within the high-risk infection cluster. From May to November 2018, faecal samples were collected from 477 dogs aged ≥6 months that visited 12 off-leash dog parks in the Halton, Hamilton and Niagara public health units. Faecal samples were analysed via a magnetic capture probe DNA extraction and real-time PCR method for E. multilocularis DNA. Overall, 0% (97.5% CI: 0%-0.80%) of samples tested positive. This result informs preventive recommendations for E. multilocularis infections in dogs in this region.

Combination of FAIMS, Protein A Depletion, and Native Digest Conditions Enables Deep Proteomic Profiling of Host Cell Proteins in Monoclonal Antibodies.

Host cell proteins (HCPs) are residual impurities generated by the expression cell line during the production of biopharmaceuticals. Although the majority of these contaminants are removed during purification, HCPs can represent a considerable risk to the efficacy and safety of a therapeutic protein if not actively monitored. The enzyme-linked immunosorbent assay (ELISA) is commonly used throughout production to monitor HCP levels but has limited ability to identify novel HCPs or provide detailed quantification. Liquid chromatography tandem mass spectrometry (LC-MS2) methods are increasingly being used in conjunction with established ELISA techniques to provide rapid adaptability to increasingly complex samples as well as highly quantitative and informative results. However, MS-based methods are still hindered by the large dynamic range between high abundance biopharmaceutical proteins and low abundance HCPs. Here, we propose a multifactorial approach designed to optimize HCP detection in purified monoclonal antibody samples with LC-MS2. By first depleting the sample of antibody on a protein A column, then specifically digesting HCPs while precipitating remaining antibody, and finally reducing spectral complexity through compensation voltage (CV) switching using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we identified multiple-fold more HCPs in the NIST monoclonal antibody standard than any single established mass spectrometry technique reported in the literature. Our analyses consistently identified over 600 high confidence mouse HCPs, a multifold increase over established methods, while maintaining high reproducibility.

Quantitative proteomic analysis of a genetically induced prostate inflammation mouse model via custom 4-plex DiLeu isobaric labeling.

Inflammation is involved in many prostate pathologies including infection, benign prostatic hyperplasia, and prostate cancer. Preclinical models are critical to our understanding of disease mechanisms, yet few models are genetically tractable. Here, we present a comparative quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation induced by conditional prostate epithelial IL-1ß expression. Relative quantification and sample multiplexing was achieved using custom 4-plex N,N-dimethyl leucine (DiLeu) isobaric tags and nanoflow ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Each set of 4-plex DiLeu reagents allows four urine samples to be analyzed simultaneously, providing high-throughput and accurate quantification of urinary proteins. Proteins involved in the acute phase response, including haptoglobin, inter-α-trypsin inhibitor, and α1-antitrypsin 1-1, were differentially represented in the urine of mice with prostate inflammation. Mass spectrometry-based quantitative urinary proteomics represents a promising bioanalytical strategy for biomarker discovery and the elucidation of molecular mechanisms in urological research.

In-Depth Characterization and Validation of Human Urine Metabolomes Reveal Novel Metabolic Signatures of Lower Urinary Tract Symptoms.

Lower urinary tract symptoms (LUTS) are a range of irritative or obstructive symptoms that commonly afflict aging population. The diagnosis is mostly based on patient-reported symptoms, and current medication often fails to completely eliminate these symptoms. There is a pressing need for objective non-invasive approaches to measure symptoms and understand disease mechanisms. We developed an in-depth workflow combining urine metabolomics analysis and machine learning bioinformatics to characterize metabolic alterations and support objective diagnosis of LUTS. Machine learning feature selection and statistical tests were combined to identify candidate biomarkers, which were statistically validated with leave-one-patient-out cross-validation and absolutely quantified by selected reaction monitoring assay. Receiver operating characteristic analysis showed highly-accurate prediction power of candidate biomarkers to stratify patients into disease or non-diseased categories. The key metabolites and pathways may be possibly correlated with smooth muscle tone changes, increased collagen content, and inflammation, which have been identified as potential contributors to urinary dysfunction in humans and rodents. Periurethral tissue staining revealed a significant increase in collagen content and tissue stiffness in men with LUTS. Together, our study provides the first characterization and validation of LUTS urinary metabolites and pathways to support the future development of a urine-based diagnostic test for LUTS.

Evaluation and Application of Dimethylated Amino Acids as Isobaric Tags for Quantitative Proteomics of the TGF-ß/Smad3 Signaling Pathway.

Isobaric labeling has become a widespread tool for quantitative proteomic studies. Here, we report the development and evaluation of several dimethylated amino acids as novel isobaric tags for quantitative proteomics. Four-plex dimethylated alanine (DiAla), valine (DiVal), and leucine (DiLeu) have been synthesized, sharing common features of peptide tagging and reporter ion production. DiAla and DiLeu are shown to achieve complete labeling. These two tags' impacts on peptide fragmentation and quantitation are further evaluated using HEK293 cell lysate. DiAla labeling generates more abundant backbone fragmentation whereas DiLeu labeling produces more intense reporter ions. Nonetheless, both tags enable accurate quantitative analysis of HEK293 cell proteomes. DiAla and DiLeu tags are then applied to study the TGF-ß/Smad3 pathway with four differentially treated mouse vascular smooth muscle (MOVAS) cells. Our MS data reveal proteome-wide changes of AdSmad3 as compared to the GFP control, consistent with previous findings of causing smooth muscle cell (SMC) dedifferentiation.1 Additionally, the other two novel mutations on the hub protein Smad3, Y226A, and D408H, show compromised TGF-ß/Smad3-dependent gene transcription and reversed phenotypic switch. These results are further corroborated with Western blotting and demonstrate that the novel DiAla and DiLeu isobaric tagging reagents provide useful tools for multiplex quantitative proteomics.

Isotopic N,N-Dimethyl Leucine (iDiLeu) for Absolute Quantification of Peptides Using a Standard Curve Approach.

Quantitative proteomics studies require an absolute quantification step to accurately measure changes in protein concentration. Absolute quantification using liquid chromatography-mass spectrometry (LC-MS) traditionally combines triple quadrupole instrumentation with stable isotope-labeled standards to measure protein concentrations via their enzymatically produced peptides. Chemical modification of peptides using labels like mass differential tags for relative and absolute quantification (mTRAQ) provides another route to determine protein quantities. This chapter describes a cost-effective and high-throughput chemical labeling method that utilizes five amine-reactive, isotopic N,N-dimethyl leucine (iDiLeu) reagents. These tags enable generation of four-point calibration curves in one LC-MS run to determine protein concentrations from labeled peptides. In particular, we provide a detailed workflow for protein quantification using the iDiLeu reagent that includes important considerations like labeling conditions and isotopic interference correction.

Custom 4-Plex DiLeu Isobaric Labels Enable Relative Quantification of Urinary Proteins in Men with Lower Urinary Tract Symptoms (LUTS).

The relative quantification of proteins using liquid chromatography mass spectrometry (LC-MS) has allowed researchers to compile lists of potential disease markers. These complex quantitative workflows often include isobaric labeling of enzymatically-produced peptides to analyze their relative abundances across multiple samples in a single LC-MS run. Recent efforts by our lab have provided scientists with cost-effective alternatives to expensive commercial labels. Although the quantitative performance of these dimethyl leucine (DiLeu) labels has been reported using known ratios of complex protein and peptide standards, their potential in large-scale proteomics studies using a clinically relevant system has never been investigated. Our work rectifies this oversight by implementing 4-plex DiLeu to quantify proteins in the urine of aging human males who suffer from lower urinary tract symptoms (LUTS). Protein abundances in 25 LUTS and 15 control patients were compared, revealing that of the 836 proteins quantified, 50 were found to be differentially expressed (>20% change) and statistically significant (p-value <0.05). Gene ontology (GO) analysis of the differentiated proteins showed that many were involved in inflammatory responses and implicated in fibrosis. While confirmation of individual protein abundance changes would be required to verify protein expression, this study represents the first report using the custom isobaric label, 4-plex DiLeu, to quantify protein abundances in a clinically relevant system.

Development and characterization of novel 8-plex DiLeu isobaric labels for quantitative proteomics and peptidomics.

RATIONALE: Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu. METHODS: The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the label's reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS(2) ) on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined. RESULTS: An 8-plex set of DiLeu reagents with 1 Da spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 µg peptide samples was calculated to be approximately $15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values. CONCLUSIONS: Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals.

Relative quantification of amine-containing metabolites using isobaric N,N-dimethyl leucine (DiLeu) reagents via LC-ESI-MS/MS and CE-ESI-MS/MS.

Tandem mass spectrometry (MS/MS)-based relative quantification by isobaric labeling is a useful technique to compare different metabolic expression levels in biological systems. For the first time, we have labeled primary and secondary amine-containing small molecules using 4-plex isobaric N,N-dimethyl leucine (DiLeu) to perform relative quantification. Good labeling efficiency and quantification accuracy were demonstrated with a mixture of 12 metabolite standards including amino acids and small molecule neurotransmitters. Labeling amine-containing metabolites with DiLeu reagents also enabled the separation of polar metabolites by nanoRPLC and improved the detection sensitivity by CE-ESI-MS. The 4-plex DiLeu labeling technique combined with LC-MS/MS and CE-MS/MS platforms were applied to profile and quantify amine-containing metabolites in mouse urine. The variability of concentrations of identified metabolites in urine samples from different mouse individuals was illustrated by the ratios of reporter ion intensities acquired from online data-dependent analysis.