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.

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.

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.

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.

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.

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.

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.

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.

Novel isotopic N,N-dimethyl leucine (iDiLeu) reagents enable absolute quantification of peptides and proteins using a standard curve approach.

Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N,N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

Comparison of NIMS and MALDI platforms for neuropeptide and lipid mass spectrometric imaging in C. borealis brain tissue.

Nanostructure-initiator mass spectrometry (NIMS) is a recently developed matrix-free laser desorption/ionization technique that has shown promise for peptide analyses. It is also useful in mass spectrometric imaging (MSI) studies of small molecule drugs, metabolites, and lipids, minimizing analyte diffusion caused by matrix application. In this study, NIMS and matrix-assisted laser desorption/ionization (MALDI) MSI of a crustacean model organism Cancer borealis brain were compared. MALDI was found to perform better than NIMS in these neuropeptide imaging experiments. Twelve neuropeptides were identified in MALDI MSI experiments whereas none were identified in NIMS MSI experiments. In addition, lipid profiles were compared using each ionization method. Both techniques provided similar lipid profiles in the m/z range 700 - 900.

From pixel to voxel: a deeper view of biological tissue by 3D mass spectral imaging.

Three dimensional mass spectral imaging (3D MSI) is an exciting field that grants the ability to study a broad mass range of molecular species ranging from small molecules to large proteins by creating lateral and vertical distribution maps of select compounds. Although the general premise behind 3D MSI is simple, factors such as choice of ionization method, sample handling, software considerations and many others must be taken into account for the successful design of a 3D MSI experiment. This review provides a brief overview of ionization methods, sample preparation, software types and technological advancements driving 3D MSI research of a wide range of low- to high-mass analytes. Future perspectives in this field are also provided to conclude that the outlook for 3D MSI is positive and promises ever-growing applications in the biomedical field with continuous developments of this powerful analytical tool.