High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry.

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.

Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

Localization of Multiple O-Linked Glycans Exhibited in Isomeric Glycopeptides by Hot Electron Capture Dissociation.

We describe a method to obtain a comprehensive profile of multiple glycosylations in glycopeptide isoforms. We detected a wide range of abundances of various O-glycoforms in isomeric glycopeptides using hot electron capture dissociation (hot ECD) in liquid chromatography-tandem mass spectrometry. To capture low abundant glycosylated species, a prototype of a ZenoTOF 7600 system incorporating an efficient electron-activated dissociation device to perform hot ECD was operated in targeted or scheduled high-resolution multiple reaction monitoring workflows. In addition, Zeno trap pulsing was activated to enhance the sensitivity of the time-of-flight mass spectrometer. Sixty-nine O-glycopeptides of the long O-glycopeptides in tryptic bovine fetuin digest were obtained with a relative abundance range from 100 to 0.2%, which included sialylated glycans with Neu5Ac and Neu5Gc.

LC-HRMS quantitation of intact antibody drug conjugate trastuzumab emtansine from rat plasma.

AIM: Compared with small molecules, LC-MS quantitation of larger biotherapeutic proteins such as antibodies and antibody-drug conjugates at the intact level presents many challenges in both LC and MS due to their higher molecular weight, bigger size, structural complexity and heterogeneity. RESULTS & CONCLUSION: In this study, quantitation of an intact lysine-linked antibody-drug conjugate, trastuzumab emtansine is presented. Trastuzumab emtansine was extracted from rat plasma using bead-based immunoaffinity capture; after elution from the beads, it was directly analyzed on a LC-HRMS system. Quantitation using both extracted ion chromatogram and deconvoluted mass peaks was evaluated. A limit of quantitation was approximately 20 ng on column with a linear dynamic range from 5 to 100 µg/ml. In addition, the reproducibility and distribution of the drug-to-antibody ratio at different trastuzumab emtansine concentrations were discussed.

Metabolomic spectral libraries for data-independent SWATH liquid chromatography mass spectrometry acquisition.

High-quality mass spectral libraries have become crucial in mass spectrometry-based metabolomics. Here, we investigate a workflow to generate accurate mass discrete and composite spectral libraries for metabolite identification and for SWATH mass spectrometry data processing. Discrete collision energy (5-100 eV) accurate mass spectra were collected for 532 metabolites from the human metabolome database (HMDB) by flow injection analysis and compiled into composite spectra over a large collision energy range (e.g., 10-70 eV). Full scan response factors were also calculated. Software tools based on accurate mass and predictive fragmentation were specially developed and found to be essential for construction and quality control of the spectral library. First, elemental compositions constrained by the elemental composition of the precursor ion were calculated for all fragments. Secondly, all possible fragments were generated from the compound structure and were filtered based on their elemental compositions. From the discrete spectra, it was possible to analyze the specific fragment form at each collision energy and it was found that a relatively large collision energy range (10-70 eV) gives informative MS/MS spectra for library searches. From the composite spectra, it was possible to characterize specific neutral losses as radical losses using in silico fragmentation. Radical losses (generating radical cations) were found to be more prominent than expected. From 532 metabolites, 489 provided a signal in positive mode [M+H]+ and 483 in negative mode [M-H]-. MS/MS spectra were obtained for 399 compounds in positive mode and for 462 in negative mode; 329 metabolites generated suitable spectra in both modes. Using the spectral library, LC retention time, response factors to analyze data-independent LC-SWATH-MS data allowed the identification of 39 (positive mode) and 72 (negative mode) metabolites in a plasma pool sample (total 92 metabolites) where 81 previously were reported in HMDB to be found in plasma. Graphical abstract Library generation workflow for LC-SWATH MS, using collision energy spread, accurate mass, and fragment annotation.

SWATH-ID: An instrument method which combines identification and quantification in a single analysis.

Data-independent acquisition (DIA) approaches, such as SWATH® -MS, are showing great potential to reliably quantify significant numbers of peptides and proteins in an unbiased manner. These developments have enhanced interest in developing a single DIA method that integrates qualitative and quantitative analysis, eliminating the need of a prebuilt library of peptide spectra, which are created through data-dependent acquisition methods or from public repositories. Here, we introduce a new DIA approach, referred to as "SWATH-ID," which was developed to allow peptide identification as well as quantitation. The SWATH-ID method is composed of small Q1 windows, achieving better selectivity and thus significantly improving high-confidence peptide extractions from data files. Furthermore, the SWATH-ID approach transmits precursor ions without fragmentation as well as their fragments within the same SWATH acquisition period. This provides a single scan that includes all precursor ions within the isolation window as well as a record of all of their fragment ions, substantially negating the need for a survey scan. In this way all precursors present in a small Q1 window are associated with their fragment ions, improving the identification specificity and providing a more comprehensive and in-depth view of protein and peptide species in complex samples.

A workflow for absolute quantitation of large therapeutic proteins in biological samples at intact level using LC-HRMS.

AIM: The commonly used LC-MS workflow to quantify protein therapeutics in biological samples is 'bottom-up' approach. In this study, the aim is to establish 'top-down' approach for absolute quantitation of therapeutic antibodies or proteins of similar sizes in biological samples at intact level. MATERIALS & METHODS: Using a recombinant human monoclonal antibody as the model molecule, we present a workflow to measure large therapeutic proteins in plasma at intact level based on deconvoluted high-resolution MS (HRMS) peaks. A novel MultiQuant™ software function was developed to automatically deconvolute the peaks and process the data. RESULTS & CONCLUSION: The workflow showed satisfying performance. This is a proof of concept study demonstrating the feasibility of bioanalysis of large therapeutic proteins at intact level using LC-HRMS.

Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins.

We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling that improves S/N by twofold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, normalized group area ratio, MLR normalization, weighted regression analysis, and data dissemination through the Yale protein expression database. As a proof of principle we developed a robust 90 min LC-MRM assay for mouse/rat postsynaptic density fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay.

Rapid characterization of naphthenic acids using differential mobility spectrometry and mass spectrometry.

To analyze the naphthenic acid content of environmental waters quickly and efficiently, we have developed a method that employs differential mobility spectrometry (DMS) coupled to mass spectrometry (MS). This technique combines the benefits of infusion-based MS experiments (parallel, on-demand access to individual components) with DMS's ability to provide liquid chromatography-like separations of isobaric and isomeric compounds in a fraction of the time. In this study, we have applied a DMS-MS workflow to the rapid gas-phase separation of naphthenic acids (NAs) within a technical standard and a real-world oil sands process-affected water (OSPW) extract. Among the findings provided by this workflow are the rapid characterization of isomeric NAs (i.e., same molecular formulas) in a complex OSPW sample, the ability to use DMS to isolate individual NA components (including isomeric NAs) for in-depth structural analyses, and a method by which NA analytes, background ions, and dimer species can be characterized by their distinct behaviors in DMS. Overall, the profiles of the NA content of the technical and OSPW samples were consistent with published values for similar samples, such that the benefits of DMS technology do not detract from the workflow's accuracy or quality.

Comprehensive analytical strategy for biomarker identification based on liquid chromatography coupled to mass spectrometry and new candidate confirmation tools.

A comprehensive analytical LC-MS(/MS) platform for low weight biomarkers molecule in biological fluids is described. Two complementary retention mechanisms were used in HPLC by optimizing the chromatographic conditions for a reversed-phase column and a hydrophilic interaction chromatography column. LC separation was coupled to mass spectrometry by using an electrospray ionization operating in positive polarity mode. This strategy enables us to correctly retain and separate hydrophobic as well as polar analytes. For that purpose artificial model study samples were generated with a mixture of 38 well characterized compounds likely to be present in biofluids. The set of compounds was used as a standard aqueous mixture or was spiked into urine at different concentration levels to investigate the capability of the LC-MS(/MS) platform to detect variations across biological samples. Unsupervised data analysis by principal component analysis was performed and followed by principal component variable grouping to find correlated variables. This tool allows us to distinguish three main groups whose variables belong to (a) background ions (found in all type of samples), (b) ions distinguishing urine samples from aqueous standard and blank samples, (c) ions related to the spiked compounds. Interpretation of these groups allows us to identify and eliminate isotopes, adducts, fragments, etc. and to generate a reduced list of m/z candidates. This list is then submitted to the prototype MZSearcher software tool which simultaneously searches several lists of potential metabolites extracted from metabolomics databases (e.g., KEGG, HMDB, etc) to propose biomarker candidates. Structural confirmation of these candidates was done off-line by fraction collection followed by nanoelectrospray infusion to provide high quality MS/MS data for spectral database queries.

Dimensionality reduction and visualization in principal component analysis.

Many modern applications of analytical chemistry involve the collection of large megavariate data sets and subsequent processing with multivariate analysis techniques (MVA), two of the more common goals being data analysis (also known as data mining and exploratory data analysis) and classification. Classification attempts to determine variables that can distinguish known classes allowing unknown samples to be correctly assigned, whereas data analysis seeks to uncover and understand or confirm relationships between the samples and the variables. An important part of analysis is visualization which allows analysts to apply their expertise and knowledge and is often easier for the samples than the variables since there are frequently far more of the latter. Here we describe principal component variable grouping (PCVG), an unsupervised, intuitive method that assigns a large number of variables to a smaller number of groups that can be more readily visualized and understood. Knowledge of the source or nature of the variables in a group allows them all to be appropriately treated, for example, removed if they result from uninteresting effects or replaced by a single representative for further processing.

Instrumental and experimental effects in LC-MS-based metabolomics.

The experimental complexity of a metabolomics study can cause uncontrolled variance that is not related to the biological effect being studied and may distort or obscure the data analysis. While some sources can be controlled with good experimental techniques and careful sample handling, others are inherent in the analytical technique used and cannot easily be avoided. We discuss the sources and appearance of some of these artifacts and show ways in which they can be detected using visualization and statistical tools, allowing appropriate treatment prior to multivariate analysis (MVA).

Investigation of analytical variation in metabonomic analysis using liquid chromatography/mass spectrometry.

Sources of analytical variation in high-performance liquid chromatography/mass spectrometry (HPLC/MS), such as changes in retention, mass accuracy or signal intensity, have been investigated to assess their importance as a variable in the metabonomic analysis of human urine. In this study chromatographic retention and mass accuracy were found to be quite reproducible with the most significant source of analytical variation in the data sets obtained being the result of changes in detector response. Depending on the signal intensity threshold used to define the presence of a peak a sample component could be present in some replicate injections and absent in others within the same run. The implementation of a more sophisticated data software analysis package was found to greatly reduce the impact of detector response variability resulting in improved data analysis.

Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.

Direct profiling of total lipid extracts on a hybrid LTQ Orbitrap mass spectrometer by high-resolution survey spectra clusters species of 11 major lipid classes into 7 groups, which are distinguished by their sum compositions and could be identified by accurately determined masses. Rapid acquisition of survey spectra was employed as a "top-down" screening tool that, together with the computational method of principal component analysis, revealed pronounced perturbations in the abundance of lipid precursors within the entire series of experiments. Altered lipid precursors were subsequently identified either by accurately determined masses or by in-depth MS/MS characterization that was performed on the same instrument. Hence, the sensitivity, throughput and robustness of lipidomics screens were improved without compromising the accuracy and specificity of molecular species identification. The top-down lipidomics strategy lends itself for high-throughput screens complementing ongoing functional genomics efforts.

Metabonomics and biomarker discovery: LC-MS metabolic profiling and constant neutral loss scanning combined with multivariate data analysis for mercapturic acid analysis.

In the field of metabonomics, 1H NMR and full scan mass spectrometry methods have usually been combined with principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to detect patterns in biofluids that correspond to specific effects, usually a toxic site effect of a compound. Confounders together with great interindividual variation complicate such analysis in humans, and therefore, metabonomic data are almost restricted to animals. In our study, a constant neutral loss (CNL) scan on a linear ion trap demonstrated increased sensitivity and specificity compared to a full scan approach and was performed to detect mercapturic acids (MA), a class of effect markers. The method was applied to human volunteers administered 50 and 500 mg of acetaminophen (AAP), a model compound known to form MAs. Using a new algorithm to prepare the CNL data for chemometrics, discrimination of control and postdose samples could be performed using PCA and PLS-DA. The loadings plots clearly revealed AAP-MA as a marker, even at low-dose levels. Orthogonal signal correction (OSC) was carried out to investigate background information that is not due to exposure. Surprisingly, the OSC data provided a classification of male and female subjects showing the performance of the new approach.

Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition.

Data-dependent acquisition of MS/MS spectra from lipid precursors enables to emulate the simultaneous acquisition of an unlimited number of precursor and neutral loss scans in a single analysis. This approach takes full advantage of rich fragment patterns in tandem mass spectra of lipids and enables their profiling by complex (Boolean) scans, in which masses of several fragment ions are considered within a single logical framework. No separation of lipids is required, and the accuracy of identification and quantification is not compromised, compared to conventional precursor and neutral loss scanning.