Accelerating covalent binding studies: Direct mass shift measurement with acoustic ejection and TOF-MS

In this report, we detail direct inject mass spectrometry via a Robotically Automated Vacuum Enabled (RAVE) interface that utilizes commercially available capillary electrophoresis hardware to directly inject samples for mass spectrometry (MS) at a sampling rate of approximately 12s per sample. This system enables direct electrospray ionization from standard 48, 96 or 384-well plates with minimal investment in hardware and software, utilizing a custom developed web app interface that provides autosampler control and analysis of raw extracted data from the mass spectrometer. We show a high level of correlation among results obtained with RAVE coupled MS, acoustic ejection (Echo) MS, and liquid chromatography coupled MS (LCMS) on 384 biocatalytically driven reactions. We additionally utilize RAVE MS on an array of 96 chemocatalytic reaction conditions to show that, while direct MS analysis can be challenging in complex mixtures, simple dilution followed by direct injection is often sufficient for analysis. With these results, we demonstrate the potential for RAVE MS to be utilized as a low-cost, low barrier to entry tool for rapid direct-inject MS analysis.

A generic high-throughput liquid chromatography (HTLC) tandem mass spectrometry (MS/MS) assay for the determination of compound I in human urine and dialysate (hemodialysis) was developed and validated. By using the HTLC on-line extraction technique, sample pretreatment was not necessary. The sample was directly injected onto a narrow bore large particle size extraction column (50 x 1.0 mm, 60 microm) where the sample matrix was rapidly washed away using a high flow rate (5 mL/min) aqueous mobile phase while analytes were retained. The analytes were subsequently eluted from the extraction column onto an analytical column using an organic-enriched mobile phase prior to mass spectrometric detection. The analytes were then eluted from the analytical column to the mass spectrometer for the determination. The linear dynamic range was 2.0-6000 ng/mL for the urine assay and 0.1-300 ng/mL for the dialysate assay. Intraday accuracy and precision were evaluated by analyzing five replicates of calibration standards at all concentrations used to construct the standard curve. For the urine assay, the precision (RSD%, n=5) ranged from 1.9 to 8.0% and the accuracy ranged from 87.8 to 105.2% of nominal value. For the dialysate assay, the precision (RSD%, n=5) ranged from 1.1 to 10.0% and the accuracy from 94.5 to 105.2% of nominal value. In-source fragmentation of the acyl glucuronide metabolite (compound III) did not interfere with the determination of parent compound I. The developed HTLC/MS/MS methodology was specific for compound I in the presence of compound III. Column life-time is increased and sample analysis time is decreased over traditional reversed-phase methods when direct injection assays for urine and dialysate are coupled with the technology of HTLC.

Acetanilide herbicides are widely used throughout the United States to control annual grasses in corn, soybeans and other crops. The herbicides studied, include the acetanilides; acetochlor, alachlor, metolachlor, propachlor and the acetamide; dimethenamid. The biological degradation of these herbicides produces a myriad of polar metabolites the most prominent of which are the ethanesulfonic acids (ESA) and oxanilic acids (OX) moieties. These degradates have been found in surface waters from agricultural runoff and in shallow groundwater due to leaching through vulnerable soils. Several less significant degradates have recently been identified and incorporated into a single multi-residue methodology. A high throughput method has been developed for the analysis of fourteen soil degradates of the specified herbicides. The method uses direct aqueous injection (DAI) liquid chromatography / mass spectrometry / mass spectrometry (LC/MS/MS) to analyze these materials without any sample pretreatment or concentration. MS/MS provides a very specific and highly sensitive mode of detection using TurboIonSpray in the negative ion mode. Thirteen of the fourteen degradates were validated at the 0.05 µg/L level in three different water matrices, raw surface water, finished surface water (drinking water) and groundwater.

Background Leakage of Cerebrospinal Fluid (CSF) can result from trauma, lacerations, and surgery, and if untreated can result in potentially life-threatening meningitis. CSF leaks can be diagnosed through the detection of β2-Transferrin (β2-Tf) from nasal secretions via gel electrophoresis. β2-Tf is a uniquely glycosylated proteoform of Transferrin found primarily in CSF, which can be distinguished from the most abundant Transferrin proteoform (β1-Tf) by its altered electrophoretic mobility. However, gel-based detection of β2-Tf requires hours of labor and has low specificity due to the numerous alternative glycoforms of Transferrin. Higher-throughput assays are thus desirable for improved patient care. Recently, our group applied High-Resolution Mass Spectrometry (HR-MS) coupled to Microprobe In-Emitter Elution (MPIE) to discriminate Transferrin isoforms. This method successfully resolved β2- from β1-Tf in nasal secretions derived from patient samples with suspected CSF leakage, but has relatively low sample-to-sample throughput. Herein, we describe a novel approach utilizing Liquid Chromatography Mass Spectrometry (LC-MS) to rapidly profile Transferrin glycoforms from patient nasal secretion samples. Furthermore, we explore solutions to address significant blood contamination in nasal secretions, which may otherwise interfere with this approach. Furthermore, we explore solutions to address significant blood contamination in some nasal secretions, which may otherwise interfere with this approach. Methods Our approach consists of two primary steps: 1) Sample processing and high abundance protein depletion via commercial filtration and depletion columns. 2) LC-MS analysis of the resulting extract to resolve Transferrin glycoforms by retention time and m/z. This approach retains the high specificity of the previously demonstrated MPIE-MS method, but takes advantage of existing high-throughput liquid chromatography platforms to reduce the sample-to-sample analysis time. Secretion samples were analyzed using a Vanquish HPLC system coupled to a Q-Exactive Plus Mass Spectrometer (ThermoFisher). Broadband mass spectra were collected (FWHM resolution of 17,500 at 200 m/z), and the deconvoluted average masses were used to identify Transferrin glycoforms. Results Our approach was benchmarked using remnant nasal secretion samples from patients with known CSF leakage. Examining 9 samples, the resulting ESI spectra revealed peaks at 79,554 Da and 78,008 Da, consistent with β1-Tf (Pred. MW (Average): 79,554.71 Da) and β2-Tf (Pred. MW (Average): 78,008.35 Da) within ∼10 ppm mass accuracy. The 79.5 kD peak was observed in all samples, while the 78 kD peak was observed in 7 of 9 samples. Samples with missing β2-Tf were marked by significant blood contamination. Genetic variants (2/9 samples) resulted in mass shifts for both β1- and β2-Tf variants, but Transferrin glycoforms identifiable due to the preserved mass shift between the β1- and β2-Tf peaks (∼1546.4 Da). An optimized LC-MS gradient enables rapid (∼10 minute) sample-to-sample injection times. Samples with blood contamination represent an additional challenge for identification of β2-Tf, owing to ion suppression from blood-derived β1-Tf. We have developed enrichment and depletion methods to increase the sensitivity for β2-Tf in blood contaminated samples, and anticipate that these approaches will reduce or eliminate this challenge. Conclusions Our approach enables high-throughput, MS-based measurement of Transferrin glycoforms (i.e. β1- and β2-Tf) from clinical nasal secretion samples for the first time.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

Structural annotation of small molecules in biological samples remains a key bottleneck in untargeted metabolomics, despite rapid progress in predictive methods and tools during the past decade. Liquid chromatography–tandem mass spectrometry, one of the most widely used analysis platforms, can detect thousands of molecules in a sample, the vast majority of which remain unidentified even with best-of-class methods. Here we present LC-MS2Struct, a machine learning framework for structural annotation of small-molecule data arising from liquid chromatography–tandem mass spectrometry (LC-MS2) measurements. LC-MS2Struct jointly predicts the annotations for a set of mass spectrometry features in a sample, using a novel structured prediction model trained to optimally combine the output of state-of-the-art MS2 scorers and observed retention orders. We evaluate our method on a dataset covering all publicly available reversed-phase LC-MS2 data in the MassBank reference database, including 4,327 molecules measured using 18 different LC conditions from 16 contributors, greatly expanding the chemical analytical space covered in previous multi-MS2 scorer evaluations. LC-MS2Struct obtains significantly higher annotation accuracy than earlier methods and improves the annotation accuracy of state-of-the-art MS2 scorers by up to 106%. The use of stereochemistry-aware molecular fingerprints improves prediction performance, which highlights limitations in existing approaches and has strong implications for future computational LC-MS2 developments.

Summary of United States Human Proteome Organisation (HUPO) Symposium Entitled “Standardized Clinical Proteomics Platforms”

Comparison of sample preparation methods, validation of an UPLC–MS/MS procedure for the quantification of cyclosporine A in whole blood sample

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.

A new method was developed for the analysis of natural and synthetic androgenic steroids and their selected metabolites in aquatic environmental matrixes using direct large-volume injection (LVI) high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). Method accuracy ranged from 87.6 to 108% for analytes with well-matched internal standards. Precision, quantified by relative standard deviation (RSD), was less than 12%. Detection limits for the method ranged from 1.2 to 360 ng/L. The method was demonstrated on a series of 1 h composite wastewater influent samples collected over a day with the purpose of assessing temporal profiles of androgen loads in wastewater. Testosterone, androstenedione, boldenone, and nandrolone were detected in the sample series at concentrations up to 290 ng/L and loads up to 535 mg/h. Boldenone, a synthetic androgen, had a temporal profile that was strongly correlated to testosterone, a natural human androgen, suggesting its source may be endogenous. An analysis of the sample particulate fraction revealed detectable amounts of sorbed testosterone and androstenedione. Androstenedione sorbed to the particulate fraction accounted for an estimated 5 to 7% of the total androstenedione mass.

The open port interface (OPI) coupled to an atmospheric pressure ion source is used to capture, dilute, focus, and transport nanoliter volume sample droplets for high-speed mass spectrometric analysis. For typical applications, the system has been optimized to achieve 1 Hz nanoliter volume sample transfer rates while simultaneously diluting the sample >1000-fold to minimize sample matrix-induced ionization suppression. Geometric, flow, and dispensing alterations to the system presented here demonstrate that sample transfer rates for the OPI of at least 15 Hz are possible. The fluid dynamic processes that enable sampling rates of 1 Hz and greater are examined in detail by correlating computational fluid dynamics simulations, analytic calculations, experimental data, photographic footage, and reference to the fluid dynamics literature. The resulting models and experimental results provide the rationale underlying the design and tuning of the system as well as information for developing optimized analytical methods. In combination with acoustic droplet dispensing, referred to as acoustic ejection mass spectrometry (AEMS), this system can be considered to be a special case of flow injection analysis with unique features that control the peak width, symmetry, and segregation of the samples transported in a fluid while simultaneously enabling their mixing and dilution with carrier fluids. In addition, conditions are established to prevent direct contact of the sample with a surface enabling, in combination with a contact-free dispenser like acoustic ejection, a dramatic reduction in sample-to-sample carry-over.

Carboxylic enantiomers are prevalent in living organisms and synthetic samples that exhibit important biological properties. The profiling of carboxylic enantiomers is beneficial for monitoring physiological states and further unraveling the metabolism mechanisms between carboxylic enantiomers and diseases. In this study, pairs of light and heavy isotope reagents, (S)-(3-aminopiperidin-1-yl) phenyl-methanone/d5-(S)-(3-aminopiperidin-1-yl) phenyl-methanone (APMA/d5-APMA), were synthesized and applied to tag the enantiomers of carboxylic metabolites and drugs. The tagging reagents of APMA and d5-APMA carry chiral amine groups that can efficiently and selectively react with the carboxyl group on carboxylic enantiomers. Upon the tagging strategy, pairs of carboxylic enantiomers showed satisfactory chromatographic performance on the conventional reversed-phase (RP) column, and the detection sensitivities of mass spectrometry (MS) analysis were also elevated. The obtained limits of detection (LODs) of 16 carboxylic enantiomers were in the range of 0.008-0.08 ng mL-1. With the developed method of APMA/d5-APMA tagging-assisted liquid chromatography-mass spectrometry (LC-MS) analysis, we successfully achieved the sensitive detection of carboxylic enantiomers in trace amounts of serum samples. The results showed that the levels of 6 carboxylic enantiomers in hepatic fibrosis serum presented significant alteration compared with normal serum samples. This proposed method provides great potential for uncovering the biological function of carboxylic enantiomers in the development of diseases.

This chapter presents an overview about the principles and instrumentation of atmospheric pressure ionization mass spectrometry (MS), followed by the application of electrospray ionization (ESI) MS and liquid chromatography/MS (LC/MS) to oligomers and polymers. A catalyst to the development of techniques involving sprays as methods of sample introduction and/or ionization has been the pursuit of on-line coupling of LC/MS. The advantage of hyphenated LC/MS for obtaining information about chemical composition, resolution of overlapping charge envelopes in the electrospray ionization mass spectra of polymers, size-exclusion chromatography calibration, and complex mixture analysis have been highlighted. Thermospray was an earlier method developed as an LC/MS interface that employed heat instead of an electric field for generation of charged droplets. ESI MS has become the method of choice in general, when on-line LC/MS of various synthetic polymers is considered. Spraying in ESI is conducted at atmospheric pressure and ambient temperature.

Mengkrengan (Polygonum barbatum L.) is a plant that is empirically used by the Dayak tribe in Kapuas Village, Central Kalimantan, Indonesia, for the treatment of typhoid fever. Secondary metabolites found in the leaves of mengkrengan include saponins, terpenoids, flavonoids, tannins, and alkaloids. The objective of this study was to determine the antibacterial activity and identify the metabolite compounds from the ethyl acetate fraction of mengkrengan (Polygonum barbatum L.) leaves originating from Central Kalimantan, Indonesia, using the Liquid Chromatography – Mass Spectrometry (LC-MS) method. The stages of the research began with the preparation of simplicia, the production of the ethyl acetate fraction, antibacterial activity testing at concentrations of 5%, 10%, and 15%, and the identification of metabolite compounds from the ethyl acetate fraction of mengkrengan leaves using LC-MS. The results obtained at concentrations of 5%, 10%, and 15% showed antibacterial activity with inhibition zones of 9 mm (moderate category), 12 mm (strong category), and 17 mm (strong category), respectively. LC-MS identification of compounds revealed a metabolite compound with the molecular formula C15H10O7 (MW 303.0511), which was identified as quercetin. The LC-MS chromatogram also showed the presence of six other unidentified compounds.

Disclosure: M.G. Hamilton: None. G. Patel: None. S. Teja Sathi: None. K. Win: None. P. Lekprasert: None. Serum thyroglobulin (Tg) levels are affected by the presence of anti-thyroglobulin antibodies (anti-Tg Abs) during surveillance of differentiated thyroid cancer (DTC). About 20% of patients have positive anti-Tg Abs post-thyroidectomy and laboratory investigations for DTC surveillance may lead to false negative results. Liquid chromatography-tandem mass spectrometry (LCMS) is superior to immunoassay in measuring Tg levels in patients with the presence of anti-Tg Abs. Mass spectrometry is the only currently available method that could eliminate the interference of anti-Tg Abs in the quantification of Tg but is not readily available. We present the case of a patient who had a recurrence of DTC despite persistently undetectable Tg levels using both immunoassay and LCMS methods. The patient is a 49-year-old female with a history of papillary thyroid cancer who underwent total thyroidectomy followed by adjuvant radio-ablation. She presented to our endocrinology office for routine follow up for her DTC. She reported subjective unknown weight loss over four months. Her physical exam was unremarkable with no palpable neck swelling or mass. Vital signs were within normal limits. She continued to have undetectable levels of Tg as measured by immunoassay, stable levels of anti-Tg Abs ranging from 19 IU/mL to 33 IU/mL, and negative findings on her thyroid ultrasound for the first four years post-op. When she presented to the office five years post-thyroidectomy her anti-Tg Ab level had increased to 114 IU/mL and subsequently to 137 IU/mL a few months later despite undetectable Tg levels on both immunoassay and LCMS. A thyroid ultrasound was completed due to rising anti-Tg Abs levels which showed a suspicious one cm mass at the right thyroid bed and suspicious bilateral cervical lymph nodes. Repeated FNA biopsy of the bilateral cervical lymph nodes confirmed follicular epithelium with nuclear inclusions and grooves, compatible with recurrent papillary thyroid carcinoma. This patient had a recurrence of her DTC despite undetectable Tg levels on both immunoassay and LCMS. Tg is an important aspect of surveillance post-treatment for all patients with DTC because its presence is a surrogate for the presence of thyroid tissue and therefore the recurrence of thyroid malignancy. Clinicians must have a high index of suspicion when monitoring a patient with a history of DTC and the presence of anti-Tg Abs because it interferes with the accurate measurement of Tg levels. A study conducted by Azmat et al. demonstrated that measuring Tg levels via LCMS has been found to eliminate the confounding effect of anti-Tg Abs with a sensitivity and specificity of 44.4% and 94.1% respectively. However, our case illustrates that even LCMS is not always reliable in such situations, and increasing levels of anti-Tg Abs should be an individual risk factor for suspecting recurrence of DTC. Presentation: 6/3/2024