Solvent effects on proteins serve as intrinsic determinants of their folding conformation, dynamic behavior, and molecular recognition. This study systematically analyzed the effects of four polar solvents, including dimethylformamide (DMF), dimethylacetamide (DMA), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO, with elevated polarities), on three prototype proteases by ion mobility mass spectrometry (IM-MS). The results demonstrated that changes in solvent type and concentration could significantly alter the gas-phase conformation of proteases, inducing varying degrees of partial unfolding and the formation of intermediate states. DMF, NMP, and DMA exhibited no significant denaturing effect on protein conformations. In contrast, DMSO with the highest polarity tended to form folded states at lower concentrations and unfolded states at higher concentrations. Further analysis revealed that the trend of collision cross-section (CCS) changes did not fully align with the patterns of the charge state, suggesting that solvent environments may regulate protein conformational diversity and charge distribution through distinct mechanisms. DMSO has been demonstrated to promote the adoption of unfolded conformations by proteins that go beyond their native state. In contrast, the other three solvents have been shown to drive the protein conformation closer to its native state without inducing excessive denaturation. This study not only reveals the influence of polar solvents on the stability and kinetics of proteases but also provides a new experimental basis for understanding the protein-solvent interaction.
Direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) has proven useful for the detection and faithful representation of labile organosulfur compounds (OSCs). Nevertheless, it has been found that when exposed to the metastable helium (He*) of the DART gas stream under soft ionization conditions, some classes of OSCs such as disulfides, undergo several reactions to produce new organosulfur species, which complicate interpretation of their spectra. In this work, these new entities were characterized and the mechanisms of their formation explored. DART-HRMS analysis of diphenyl disulfide (PhSSPh) exhibited peaks consistent with [PhSSPh]+•, [PhSSPh + H]+, [PhSPh]+•, and [PhSOSPh + H]+ and various fragments and adducts including [PhSS]+ and [Ph3S3]+. Ab initio/DFT calculations coupled with variational transition state theory revealed that several of these peaks are artifacts of reactions occurring with He* and •OH. Branching ratio analysis showed the dominant decomposition pathway of [PhSSPh]+• to be [PhSSPh]+• → PhS• + PhS:, rather than [PhSSPh]+• → PhS+=S + Ph•. The PhS•, Ph•, and •OH formed serve as key intermediates in subsequent reactions that lead to the m/z values observed. From the results, a systematic approach for the interpretation of the DART mass spectra of organic disulfides was developed and successfully applied to various disulfide compound classes (e.g., aryl, alkyl, benzyl, and phenyl). Organic solvents were also observed to influence the ability to detect the compounds. Benzene; dichloromethane; hexane; and with some exceptions, ethyl acetate and tetrahydrofuran were found to be suitable, while disulfide signals in dimethylformamide were totally absent.
Comprehensive characterization of monoclonal antibody (mAb) charge heterogeneity is essential to ensure product quality, maintain batch consistency and support biosimilar development. Charge variant analysis (CVA) is widely used to separate acidic and basic proteoforms from the main species. However, cation-exchange chromatography coupled to mass spectrometry provides limited information and cannot localize the post-translational modifications (PTMs) responsible for mAb heterogeneity. Here, the coupling of pH-gradient CVA with native top-down mass spectrometry (TD-MS) for proteoform-specific analysis of trastuzumab is presented. Individual charge variants were chromatographically separated under native conditions and directly fragmented on the chromatographic time scale using higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD) and ultraviolet photodissociation (UVPD). The addition of proton-transfer charge reduction (PTCR) helped reduce spectral congestion and enhanced the detection of high-mass fragment ions, resulting in improved sequence coverage. This workflow enabled the complete sequencing of the complementarity-determining region (CDR) 3 and the direct identification and insights into the location of key PTMs at the intact-protein level, including deamidation, succinimide and N-terminal pyroGlu for individual proteoforms. Comparison of five trastuzumab samples (originator and biosimilars) demonstrated high reproducibility in fragmentation patterns, sequence coverage and variant assignment, highlighting the robustness of the method. Although limitations remain due to the challenges of fragmenting intact mAbs under native conditions, this work establishes a proof of concept for CVA native TD-MS characterization of mAbs to complement bottom-up and middle-down analyses, and has potential for broad applicability for antibody-based biopharmaceuticals.
Top-down mass spectrometry provides a powerful approach for analyzing and quantifying intact proteoforms, i.e., the distinct molecular forms of proteins. Isobaric labeling-based quantification strategies offer the advantages of multiplexing and increased analytical depth. However, a major challenge remains the quantification of proteoforms when their precursor signals overlap, leading to mixed reporter ion intensities. In this proof-of-concept study, we employed proton transfer charge reduction (PTCR) at the MS2 level to resolve overlapping precursor signals, allowing selective isolation of individual proteoforms and subsequently, their accurate reporter ion quantification at the MS3 level. Using direct infusion mass spectrometry of model proteins labeled with cysteine-directed tandem mass tags, we demonstrate that this approach enables accurate, interference-free reporter ion-based quantification in the presence of overlapping proteoforms and spectrally congested backgrounds. This work highlights PTCR as a versatile gas-phase separation strategy to enhance the quantitative capabilities of labeling-based top-down mass spectrometry, offering a path toward precise, proteoform-resolved quantification across diverse experimental approaches, such as large-scale top-down proteomics.
This research study assesses the applicability of drift tube ion mobility spectrometry (DTIMS) to resolve ammonia (NH3) from its isotopologue, ammonia-d3 (ND3). DTIMS, known for its rapid response at ambient pressure, was employed to conduct real-time analysis of gaseous samples. Two ammonia introduction methods were evaluated, evaporation of a 3 ppm(v) aqueous ammonia solution and dry gas synthesis. The study tested these methods for the mixture analysis of ammonia and examined their impact on resolution. The resolution of isotopologues and the influence of moisture in the carrier gas on separation were assessed. Individual analysis by evaporation method provided drift times of 6.03 ms for NH3 and 6.17 ms for ND3, from which reduced mobility (K0) values of 2.52 cm2 V-1 s-1 and 2.46 cm2 V-1 s-1 were calculated, respectively. The presence of moisture in the carrier gas was found to significantly reduce the resolution of isotopologue separation. To address this limitation and enhance signal clarity, the application of wavelet denoising was conducted, with universal thresholding based on Symlet, Daubechies, and Coiflet wavelet families systematically evaluated. The Daubechies wavelet (db9) at level 9 was identified as the optimal denoising approach. Following this, multivariate curve resolution alternating least-squares (MCR-ALS) was employed for the decomposition of the complex overlapping signals into their pure ion mobility spectra and corresponding concentration profiles over time. The integration of wavelet-based denoising and MCR-ALS offers a practical strategy for the enhancement of DTIMS performance in complex isotopologue separation, particularly for future studies focused on quantitative analysis. Future work will involve the integration of steady-state isotopic transient kinetic analysis (SSITKA) for a comprehensive kinetic framework for kinetic models.
Phosphatidylcholines (PCs), which differ in fatty acyl chain lengths and degrees of unsaturation, often exist as complex mixtures of isomeric and isobaric compounds. Accurate structural identification of these lipids in imaging mass spectrometry (IMS) is essential for contextualizing their spatial distributions within tissue biochemistry. Gas-phase charge inversion ion/ion reactions offer a powerful approach to improve lipid identification by converting precursor ion types prior to dissociation, yielding more structurally informative fragmentation patterns. Herein, we employ a novel multiply charged reagent ion, 1,2,4,5-tetrakis(4-carboxylphenyl)benzene (TCPB), to perform charge inversion ion/ion reactions with protonated PC analytes. The use of higher reagent charge states improves the kinetics of the ion/ion reaction, reducing the time required for reactions to occur within an imaging experiment. Additionally, the use of higher reagent charge states results in more exothermic reactions, which facilitates consecutive fragmentation of ion/ion reaction complexes to the desired fatty acyl product ions without the need for supplemental activation, further improving the speed and efficiency of this process. This optimized workflow is applied in imaging mass spectrometry experiments to spatially map PC 34:1 isomers within rat brain tissue, revealing distinct spatial distributions for PC 16:0/18:1 and PC 18:1/16:0. These results underscore the importance of isomer resolution in lipid imaging and demonstrate the potential for exploiting reaction kinetics to improve ion/ion reaction isomer and isobar separation in imaging applications.
We explore mass-resolved imaging of fragments generated from single macromolecular assembly (MMA) ions on a custom-built Orbitrap/time-of-flight (TOF) mass spectrometer with integrated UV photodissociation (UVPD) and a position- and time-sensitive Timepix3 imaging detector assembly. We postulated that the 2D detector images provide information about the 3D geometry of the MMAs in the gas phase as the TOF analyzer has the ability to retain the relative positions of the product ions following the fragmentation process and until they reach the imaging detector, when the fragmentation occurs at the level of single-precursor MMA ion. We demonstrate that the Orbitrap/TOF mass spectrometer enables fragmentation at the single-precursor MMA ion level using dimeric and tetrameric noncovalently bound assemblies. Timepix3-derived relative position data from single-precursor fragmentation events of two distinct tetrameric MMAs reveal different higher-order structural signatures that enable their differentiation. Furthermore, mapping these single-precursor fragmentation events to possible dissociation pathways provides insight into the underlying dissociation mechanisms. Overall, this study demonstrates the potential of single-ion mass-resolved imaging to understand UVPD dissociation mechanisms, fragmentation pathways of MMA ions, and their higher-order structure.
To achieve a more accurate evaluation of the internal energy distributions of ions activated by collision-induced dissociation (CID) in the collision cell, fluorine-containing phenyl sulfates were systematically screened by calculating their dissociation energies using quantum chemical calculations at the DLPNO-CCSD(T)/CBS//ωB97X-D/def2-TZVPPD level of theory. Fluorine substitution at the ortho- and meta-positions of phenyl sulfate lowers the dissociation energy, thereby promoting dissociation. In contrast, fluorine substitution at the para position has little effect on the dissociation energy. Although para-trifluoromethyl substitution reduces the dissociation energy, it significantly decreases the dissociation rate. Notably, despite the reduced dissociation energy upon para-trifluoromethyl substitution, 4-trifluoromethyl phenyl sulfate exhibits the strongest resistance to dissociation owing to its markedly slower dissociation rate. The experimentally observed dissociation behavior showed good agreement with the dissociation energies predicted by quantum chemical calculations. These fluorine-containing phenyl sulfates, which predominantly generate a single fragment ion due to sulfur trioxide loss, were experimentally demonstrated to be suitable thermometer ions for characterizing the internal energy distributions of ions produced by CID-activated ions. By enabling systematic control of the dissociation energy through fluorine substitution patterns, this approach allows more accurate determination of internal energy distributions of CID-activated ions. The reported method provides a useful strategy for determining optimal voltages in negative electrospray ionization and subsequent tandem mass spectrometric analysis of acidic analyte molecules.
We have developed a compact, chemical ionization mass spectrometer for the sensitive detection of atmospheric trace gases using a commercial residual gas analyzer (RGA). A high-pressure ion molecule reactor interface incorporating two radiofrequency (RF)-only octupoles, powered by Wisconsin Oscillator RF power supplies, is used to focus ions through three stages of pumping before analysis via a modified RGA. We operated this instrument with benzene cation cluster reagent ions generated using a 210Po α emitter to enable the efficient ionization, transfer, and detection of analyte ions and ion-molecule clusters. Using single ion monitoring, the instrument demonstrated high sensitivity to dimethyl-1,1,1-d3 sulfide, corresponding to a 20 s 3σ limit of detection (LOD) of 18 ppt. This detection threshold is limited by the magnitude and variability in the background determination. We characterized the impact of tuning instrument pressures and ion optics on sensitivity and ion clustering to demonstrate the potential for enhanced sensitivities and electronically controllable clustering modalities. The application of the instrument to the measurement of ambient trace gases, in scan mode, was tested by sampling continuously through a window inlet on the fourth floor of the UW-Madison chemistry building (Madison, WI) for 3 days during the summer of 2025, resulting in the detection of four unique, time-correlated ammonia clusters via peak integration. The simple, robust design and relatively low cost of the instrument highlight the utility of commercial residual gas analyzers as mass analyzers for gas-phase measurements of trace species.
Glycans are essential components of cells involved in numerous biological processes, and changes in glycan profiles are often correlated with disease progression. Glycans are comprised of various monosaccharides linked together at different positions with varied stereochemistry. The structural diversity and complexity present unique analytical challenges that can limit understanding of their functional role. In addition, glycans are frequently decorated with a diverse set of chemical modifications, termed post-glycosylation modifications (PGMs). Characterization of PGMs is essential for a thorough understanding of the glycome; however, the technical challenges and low throughput of current methodologies limit our understanding of PGMs. Here, we demonstrate a novel approach for rapid visualization of specific PGMs present in tissue N-glycans by applying PGM-targeting enzymes to matrix-assisted laser desorption ionization-based mass spectrometry imaging. The method enables in situ investigation of PGMs, allowing identification of the modified sugar residue, while visualizing the spatial distribution of each modified N-glycan. As the repertoire of PGM-targeting enzymes expands, we anticipate that this approach will improve our understanding of PGM distributions within the dynamic N-glycome, providing new biological insights into the identification of novel disease biomarkers.
Hemoglobin is the protein responsible for oxygen transport in many vertebrates, and any structural alteration can lead to health issues. Although well characterized in humans, it is less known for birds. Studies have reported the identification of several α subunits along with the presence of endogenous cofactors in different avian erythrocytes. However, the native structure of avian hemoglobin remains elusive. Native top-down mass spectrometry (nTD-MS) offers powerful insights into biomolecular complexes, providing information on quaternary structure, subunit connectivity, stoichiometry, and subunit sequences. However, most nTD-MS approaches use direct infusion, which can limit the multiprotein complex population characterization. Here, we report on the development of a size-exclusion-chromatography (SEC) nTD-MS approach, including different activation methods to characterize the zebra finch hemoglobin structure. Three different tetramer populations were separated and characterized using optimized pMS2 (pseudo-MS2, complex-up) workflows to induce subunit and endogenous cofactor release previously reported by our group. The analytical strategy was improved with the addition of an extra level of characterization through the implementation of a pMS3 (pseudo-MS3, complex-down) step with controlled pressure, allowing almost complete sequence coverage of all the subunits (>94%), along with the identification of inositol pentaphosphate as a cofactor of the tetramer structure. Altogether, these results pinpoint the key role of SEC-nTD-MS workflows to enable a complete structural characterization of hemoglobin complexes, which could provide crucial information regarding oxygen affinity, bird environment adaptation, or phylogeny.
Resolution and sensitivity are critical performance metrics for miniature ion trap mass spectrometry, collectively determining their range of applications. This study presents a novel strategy for enhancing the resolution and sensitivity of continuous atmospheric pressure interfaced ion trap mass spectrometry operating under high pressure by developing a pulsed-closure assisted continuous atmospheric pressure interface (PCA-CAPI). Ion injection and trapping were achieved under relatively high pressures, while mass analysis was conducted at significantly reduced pressures by a deliberate pulsed closure of the normally open pinch valve after ion injection. The optimal closure time of the pinch valve was systematically determined, and performance improvements across various operating pressures were evaluated. After comprehensive optimization, the resolution and intensity were increased by 4.7-fold and 5.2-fold simultaneously. Additionally, excellent ion utilization efficiency and a limit of detection of 0.5 pg/μL were achieved. High stability was obtained with a relative standard deviation of 4.33%. The capability of the PCA-CAPI-ITMS to accurately resolve and detect adjacent ions, thereby preventing potential misidentification, was validated through the analysis of two drug mixtures. The successful implementation of PCA-CAPI-ITMS offers a promising pathway toward simplified vacuum systems and opens new possibilities for the development of higher-pressure ITMS, potentially enhancing the robustness and versatility of the technique.
Drug mixture composition and compound identifications provide public health, first responder, and law enforcement communities with critical and actionable data, guiding emergency response and interdiction, informing the public, and targeting overdose prevention. The advent of novel synthetic opioids, nitazenes, and benzodiazepines, along with the spread of strong veterinary tranquilizer adulterants (e.g., xylazine and medetomidine), have created an ever-changing drug landscape. The Testing, Rapid Analysis, and Narcotic Quality (TRANQ) Research Act of 2023 directs research, method development, measurement science, and standards to address these hurdles. In conjunction with the National Institute of Standards and Technology (NIST) Rapid Drug Analysis and Research (RaDAR) program, aimed at monitoring the chemical makeup of the drug landscape, we are investigating analytical instrumentation to enable drug screening to move from the laboratory to an agile point-of-need setting. We explored compound identification with a direct analysis in real time triple quadrupole mass spectrometer (DART-TQ-MS) and the NIST/NIJ DART-MS Data Interpretation Tool with Forensics Database. Full scan analysis of single-component standards demonstrated limits of detection generally in the tens to hundreds of picograms with a few in the single nanogram range (i.e., compounds with extremes in volatility). Single-component, mixtures, and real-world street drug samples were investigated with potential identifications made matching against the high-resolution mass spectral library, using both full scan and product ion scan spectra. The development of a compact TQ-MS system enables the future potential for rapid on-site analysis, supporting public health, law enforcement, and forensic applications. This innovation paves the way for mobile, point-of-need drug testing and identification, enhancing our ability to respond to emerging drug threats.
Top-down mass spectrometry (TDMS) is a powerful platform for the structural and functional analysis of intact proteins, enabling the detailed characterization of proteoforms and precise localization of post-translational modifications. The incorporation of alternative fragmentation techniques, such as electron transfer dissociation, electron transfer higher-energy collisional dissociation, and ultraviolet photodissociation, in instruments such as Tribrid Orbitrap mass spectrometers enhances sequence coverage and improves the confidence in PTM assignment. However, tandem mass spectrometry of intact proteins >30 kDa presents substantial challenges. The resulting spectra are often highly complex with overlapping product ion signals that complicate spectral interpretation. Although increasing the mass resolution can help resolve closely spaced product ions, it is often insufficient to fully alleviate spectral congestion for large proteins. In such cases, proton transfer charge reduction (PTCR) can simplify mass spectra by dispersing product ions across a wider mass-over-charge (m/z) range. In this study, we evaluated the impact on TDMS of increasing resolving power and PTCR-enabled spectral simplification using four intact proteins: enolase (46.6 kDa), carbonic anhydrase (29 kDa), myoglobin (16.9 kDa), and ubiquitin (8.6 kDa). For carbonic anhydrase, combined MS2 fragmentation at low resolving power (60,000 at m/z 200) yielded 50.5% sequence coverage, which increased to 92.6% at high resolving power (480,000 at m/z 200) and further to 97.7% when PTCR was applied. This approach was applied to the characterization of biopharmaceuticals by analyzing the three digested and disulfide-reduced ∼25 kDa subunits of the NIST monoclonal antibody (mAb) on a liquid chromatography time scale.
Direct analysis in real time-mass spectrometry (DART-MS) is a technique well suited for screening seized drug samples as it enables rapid analysis with minimal sample preparation. Therefore, information present in full scan mass spectra is extremely important and can indicate the presence of controlled substances in a sample. This study explores the formation of oxygenated species in the full scan mass spectra of 18 nitazene analogs, as well as a few other seized drug-related compounds. Generally, with helium as the source gas, an [M+H+O-2H]+ (i.e., +14 Da) ion was present, and with nitrogen as the source gas, an ion representing [M+H+O]+ (i.e., +16 Da) was observed. However, certain compounds, such as tetracaine, contained a +16 Da ion with helium as the source gas, indicating that both the source gas and chemical properties of the analyte impact oxygenated species formation. Further tandem mass spectrometry (MS/MS) studies were conducted to identify where the oxygen adduct was formed. The presence of a tertiary amine appears to play a role in oxygenation, as oxygenated iminium ions were frequently observed. Overall, oxygenated species were observed at higher abundances for the nitazene analogs than other controlled substances, providing an additional piece of support for a nitazene analog identification using DART-MS.
We used native mass spectrometry (MS) to investigate how the architecture of the p21 DNA response element (DNA-RE) controls the binding mode of the tumor suppressor protein p53. We analyzed tetrameric full-length wild-type p53 and compared its DNA binding behavior with a dimeric variant, p53L344A. In total, 37 DNA constructs derived from p21 DNA-RE were examined via native MS, including full-site sequences, isolated half-sites, variants differing in length and composition, and random DNA sequences. The aim was to define the minimal DNA requirements for p53 binding using native MS as a robust platform for comparative analysis of p53:DNA assemblies. Our results show that flanking regions of the full 20-bp DNA-RE have no influence on the initial formation of p53:DNA complexes. However, the complete 20-bp site is required for both p53wild-type and p53L344A to bind to DNA as tetramers. Binding of a single dimer to an isolated half-site is insufficient for generating the stable tetrameric p53:DNA complex. These findings indicate that dimer-dimer interactions are crucial for stabilizing the tetrameric p53:DNA complex.
Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) is a unique, rapidly evolving technique that has been widely adopted for the characterization of the higher-order structure of proteins. Numerous statistical tools are available in the literature that can be used to identify statistically significant differences in the deuterium uptake values. Biopharmaceutical comparability studies, however, require evidence that two protein samples are highly similar and therefore necessitate a different statistical approach. Hageman et al. recently introduced an innovative HDX-MS equivalence testing method utilizing univariate two one-sided tests (TOST). The acceptance limits were established by the randomized resampling of eight replicate measurements of a reference protein. However, this approach can introduce a non-negligible level of variability in the acceptance limits when the same data set is reanalyzed. In the present study, we enhance this method by replacing the randomized resampling process with the systematic enumeration of all possible combinations of the reference data set, thus eliminating the resampling-induced variation. Because this approach incorporates all measurements, including replicate combinations with markedly elevated variability, it leads to higher acceptance limits. Therefore, we evaluated three strategies: robust outlier detection, a percentile-based method, and a partitioning approach to establish more stringent criteria and reduce patient risk. By applying the enhanced methods to data sets of three approved infliximab biosimilars and a partially deglycosylated NIST mAb used as a mock candidate biosimilar, we demonstrated correct classification of equivalent and nonequivalent samples, making the enhanced evaluation strategy well suited for regulatory comparability assessment.
Host cell proteins (HCPs) are endogenous proteins generated in cellular production systems alongside the biotherapeutic of interest. Removal of HCPs is crucial as they can be detrimental to product efficacy and patient safety. Due to its ability to determine individual HCP concentrations, liquid chromatography tandem mass spectrometry is increasingly utilized as an orthogonal method to ELISA for HCP monitoring. For protein biotherapeutics like monoclonal antibodies, their dynamic range makes detection of low-level HCPs difficult. The Orbitrap Astral MS has the potential to overcome such challenges, offering improvements in protein identifications in complex sample matrices while simultaneously reducing analysis times. Here, we utilize the Orbitrap Astral MS to perform HCP analysis on 36 protein biotherapeutics. Our workflow used a short 60 samples-per-day separation method and was initially benchmarked against four previously published studies, demonstrating comparable levels of HCP identifications. 236 HCPs were detected across the cohort and 55% of those found to be quantifiable in at least one product using label free quantitation. Functional analysis revealed that most detected HCPs had functions related to catalysis or binding, predominately catalytic activity (46%, 97 gene IDs) or protein binding (44%, 91 gene IDs). Nearly 80% of quantifiable HCPs were detected at concentrations below 10 ng/mg, with 8% detected below concentrations of 1 ng/mg. These included HCPs considered as "high-risk" by the Biophorum Development Group. This study shows how new generation mass spectrometry instruments can enable detection of low-level HCPs while allowing for a rapid and more informed understanding of a product's HCP content.
Spatial omics technologies, such as mass spectrometry imaging (MSI), can capture biomolecular distributions and their spatial locations directly from a tissue, but these distributions are not easily associated with tissue morphology without additional microscopy performed on the sample. To identify tissue regions of interest (ROIs), a reference image (such as a stained tissue, microscopy images, etc.) may be used and paired with mass spectrometry data. Due to the intense time requirements of manually labeling ROIs, segmentation models have been demonstrated as time-saving alternatives, as they automatically annotate ROIs by grouping like pixel intensities together. Supervised approaches trained on specific image types are preferred, but when those cases are not available, generalizable unsupervised segmentation models may then be used. Here, eight unsupervised semantic segmentation algorithms in R and Python, representing both statistical and machine learning algorithms, were compared for their ability to match manual annotations of 30 tiled PAS-stained kidney images and 25 tiled plant root images. Noise reduction techniques such as dimension reduction were tested to see whether they improved segmentations, and all models were applied to the full stitched images. Performance metrics were calculated to provide recommendations on the highest performing models to demonstrate their potential for automated annotation of tissue ROIs. At small cluster sizes, k-means and pytorch-tip tended to have the best performance in terms of balanced accuracy and time, though all algorithms had decreased performance at higher cluster numbers. Lastly, the segmentation model choice was demonstrated to have an impact on downstream statistics, highlighting the importance of testing and selecting the best segmentation model on a case-by-case basis, as no one model had the best performance in every comparison.
Ciprofloxacin is a widely studied fluoroquinolone antibiotic and serves here as a relevant test case for molecules exhibiting multiple protonation isomers (protomers) with abundances that vary depending on the ionization conditions using mass spectrometry (MS). Understanding the protonation behavior of such molecules during positive-ion electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) is interesting from a perspective of fundamental ion chemistry, but also crucial in achieving efficient ionization, detection, and accurate identification in an analytical context. For example, different protomers may exhibit different fragmentation patterns in tandem MS applications. While previous research has utilized MS with ion mobility spectrometry (IMS) or infrared ion spectroscopy (IRIS) to analyze ciprofloxacin, this study integrates all three techniques by performing ion mobility-selected IRIS experiments to definitively assign the protomers of ciprofloxacin and of related piperazinyl building blocks. This combined approach exposes a subtle scenario in which the type of ion source and its mode of operation influence the protomeric structures that are produced and, moreover, where the downstream ion transfer and storage stages may induce proton-migration. These findings provide guidance for the development of general workflows for mass spectrometry-based assessment of analytes with multiple protomers.