Field Asymmetric Waveform Ion Mobility Spectrometry Research Articles

Improvement of tumor neoantigen detection by high field asymmetric waveform ion mobility mass spectrometry.

Cancer neoantigens have been shown to elicit cancer-specific T-cell responses and have garnered much attention for their roles in both spontaneous and therapeutically induced antitumor responses. Mass spectrometry (MS) profiling of tumor immunopeptidomes has been used, in part, to identify MHC-bound mutant neoantigen ligands. However, under standard conditions, MS-based detection of such rare but clinically relevant neoantigens is relatively insensitive, requiring 300 million cells or more. Here, to quantitatively define the minimum detectable amounts of therapeutically relevant MHC-I and MHC-II neoantigen peptides, we analyzed different dilutions of immunopeptidomes isolated from the well-characterized T3 mouse methylcholanthrene (MCA)-induced cell line by MS. Using either data-dependent acquisition (DDA) or parallel reaction monitoring (PRM), we established the minimum amount of material required to detect the major T3 neoantigens in the presence or absence of high field asymmetric waveform ion mobility spectrometry (FAIMS). This analysis yielded a 14-fold enhancement of sensitivity in detecting the major T3 MHC-I neoantigen (mLama4) with FAIMS-PRM compared with PRM without FAIMS, allowing ex-vivo detection of this neoantigen from an individual 100 mg T3 tumor. These findings were then extended to two other independent MCA-sarcoma lines (1956 and F244). This study demonstrates that FAIMS substantially increases the sensitivity of MS-based characterization of validated neoantigens from tumors.

Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae.

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

Quantitative Time-Course Analysis of Osmotic and Salt Stress in Arabidopsis thaliana Using Short Gradient Multi-CV FAIMSpro BoxCar DIA

A major limitation when undertaking quantitative proteomic time-course experimentation is the tradeoff between depth-of-analysis and speed-of-analysis. In high complexity and high dynamic range sample types, such as plant extracts, balance between resolution and time is especially apparent. To address this, we evaluate multiple compensation voltage (CV) high field asymmetric waveform ion mobility spectrometry (FAIMSpro) settings using the latest label-free single-shot Orbitrap-based DIA acquisition workflows for their ability to deeply quantify the Arabidopsis thaliana seedling proteome. Using a BoxCarDIA acquisition workflow with a -30 -50 -70 CV FAIMSpro setting, we were able to consistently quantify >5000 Arabidopsis seedling proteins over a 21-min gradient, facilitating the analysis of ∼42 samples per day. Utilizing this acquisition approach, we then quantified proteome-level changes occurring in Arabidopsis seedling shoots and roots over 24h of salt and osmotic stress, to identify early and late stress response proteins and reveal stress response overlaps. Here, we successfully quantify >6400 shoot and >8500 root protein groups, respectively, quantifying nearly ∼9700 unique protein groups in total across the study. Collectively, we pioneer a short gradient, multi-CV FAIMSpro BoxCarDIA acquisition workflow that represents an exciting new analysis approach for undertaking quantitative proteomic time-course experimentation in plants.

Investigation of Zero-/High-Field Ion Mobility Orthogonal Separation Using a Hyphenated DMA–FAIMS System and Validation of the Two-Temperature Theory at Arbitrary Field for Tetraalkylammonium Salts in Nitrogen

Toward greater separation techniques for ions, a differential mobility analyzer (DMA) has been coupled with field asymmetric waveform ion mobility spectrometry (FAIMS) to take advantage of two mobility-related but different methods of separation. The filtering effect of the DMA allows ions to be selected individually based on low-field mobility and studied in FAIMS at variable electric field, yielding mobility separations in two dimensions. Because spectra fully describe ion mobility at variable field strength, results are then compared with a two-temperature theory-predicted mobility up to the fourth-order approximation. The comparison yields excellent results up to at least 100 Td, beyond which the theory deviates from experiments. This is attributed to two effects, the enlargement of the structure due to ion heating and the inelasticity of the collisions with the nitrogen bath gas. The corrected mobility can then be used to predict the dispersion plot through a newly developed implicit equation that circumvents the possible issues related to the more elaborate Buryakov equation. Our results simultaneously show that the DMA-FAIMS coupling yields complete information on ion mobility versus the field-strength to gas-density ratio and works toward predicting such spectra from ion structures and gas properties.

Two-step particle swarm optimization algorithm for effective deconvolution and resolution enhancement of various overlapping peaks.

The existing particle swarm optimization (PSO) algorithms are only effective in deconvoluting the overlapping peaks in ion mobility spectra with fewer than four component peaks, which limits the applicability of these algorithms. A high-performance two-step particle swarm optimization (TSPSO) algorithm was developed. Compared to the existing PSO algorithms, TSPSO can narrow the search ranges of all coefficients for the overlapping peaks through Gaussian model calculation, and thus can deconvolute various overlapping peaks with high accuracy, even for 30-component overlapping peaks. In addition, the TSPSO could be further applied to enhance the resolution of the spectra by narrowing the peak widths after the peak deconvolution. Simulated overlapping peaks were first used to evaluate the performance of TSPSO as compared to the dynamic inertia weight particle swarm optimization (DIWPSO) algorithm. The results showed that the profiles of the peaks deconvoluted by using TSPSO were more consistent with the original ones. The fitness values and the standard deviations of the fitness values from TSPSO were also at least an order of magnitude less than those from DIWPSO. By applying TSPSO, the overlapping peaks from both mass spectrometry (MS) and field asymmetric waveform ion mobility spectrometry (FAIMS) spectra can also be well deconvoluted. In addition, the resolutions of the MS and FAIMS spectra can be effectively enhanced after peak deconvolution. The enhanced spectra matched excellently with the experimental ones acquired at high-resolution modes. The experiment results convincingly demonstrate that the TSPSO algorithm is capable of both deconvoluting complex overlapping peaks and enhancing the spectrum resolution with high accuracy.

Bile-volatile organic compounds in the diagnostics of pancreatic cancer and biliary obstruction: A prospective proof-of-concept study.

Detection of volatile organic compounds (VOCs) from bodily fluids with field asymmetric waveform ion mobility spectrometry (FAIMS) and related methods has been studied in various settings. Preliminary results suggest that it is possible to detect prostate, colorectal, ovarian and pancreatic cancer from urine samples. In this study, our primary aim was to differentiate pancreatic cancer from pancreatitis and benign tumours of the pancreas by using bile samples obtained during endoscopic retrograde cholangiopancreatography (ERCP). Secondarily, we aimed to differentiate all pancreatic region malignancies from all other kinds of benign causes of biliary obstruction. A bile sample was successfully aspirated from 94 patients during ERCP in Tampere University Hospital. Hospital and patient records were prospectively followed up for at least two years after ERCP. Bile samples were analysed using a Lonestar chemical analyser (Owlstone, UK) using an ATLAS sampling system and a split-flow box. Diagnoses and corresponding data from the analyses were matched and divided into two subcategories for comparison. Statistical analysis was performed using linear discriminant analysis, support vector machines, and 5-fold cross-validation. Pancreatic cancers (n=8) were differentiated from benign pancreatic lesions (n=9) with a sensitivity of 100%, specificity of 77.8%, and correct rate of 88%. All pancreatic region cancers (n=19) were differentiated from all other kinds of benign causes of biliary obstruction (n=75) with corresponding values of 21.1%, 94.7%, and 80.7%. The sample size was too small to try to differentiate pancreatic cancers from adjacent cancers. Analysing bile VOCs using FAIMS shows promising capability in detecting pancreatic cancer and other cancers in the pancreatic area.

Development of a semi-automated volatile organic compounds (VOCs) sampling system for field asymmetric ion mobility spectrometry (FAIMS) analysis

In recent years, applications of volatile organic compounds (VOCs) sensing technologies such as field asymmetric-waveform ion-mobility spectrometry (FAIMS) system in agriculture have accelerated. FAIMS system for VOCs sensing is attractive as it offers high sensitivity, selectivity, real-time monitoring, and portability. However, the development of a robust instrumentation system is needed for precise sampling, high accumulation of VOCs, and careful handling of samples. In this study, we developed a simple semi-automated VOC sampling (SAVS) system using a Raspberry Pi microcontroller, flowmeters, electromechanical solenoid, and cellphone-based app to control cleaning and sampling loops. The system was compared with customized headspace sampling apparatus (CHSA) and validated with a biomarker (acetone) identified to be associated with potato rot development during postharvest storage. The standard error within ion current data across different compensation voltage was lower using the SAVS system than the CHSA. In addition, the maximum peak values across scans displayed a high coefficient of variation using the CHSA (16.23%) than the SAVS system (4.51%). Future work will involve improving system efficiency by adapting multiple sample units, system miniaturization, and automating the flowmeter operation. Such automation is critical to characterize VOCs precisely and automatically across several samples for multiple applications such as pathogen detection, evaluation of crop responses, etc.

Assessing the Dipole Moments and Directional Cross Sections of Proteins and Complexes by Differential Ion Mobility Spectrometry.

Ion mobility spectrometry (IMS) has become a mainstream approach to fractionate complex mixtures, separate isomers, and assign the molecular geometries. All modalities were grouped into linear IMS (based on the absolute ion mobility, K) and field asymmetric waveform IMS (FAIMS) relying on the evolution of K at a high normalized electric field (E/N) that induces strong ion heating. In the recently demonstrated low-field differential (LOD) IMS, the field is too weak for significant heating but locks the macromolecular dipoles to produce novel separations controlled by the relevant directional collision cross sections (CCSs). Here, we show LODIMS for mass-selected species, exploring the dipole alignment across charge states for the monomers and dimers of an exemplary protein, the alcohol dehydrogenase. Distinct conformational families for aligned species are revealed with directional CCS estimated from the field-dependent trend lines. We set up a model to extract the fractions of pendular conformers as a function of field intensity and translate them into dipole moment distributions. These developments make a critical step toward establishing LODIMS as a new tool for top-down proteomics and integrative structural biology.

Two-Dimensional FAIMS-IMS Characterization of Peptide Conformers with Resolution Exceeding 1000.

A high-performance field asymmetric waveform ion mobility spectrometry (FAIMS)-IMS-MS platform was developed and applied to explore the conformational diversity of the singly and doubly charged bradykinin (BK + H+)+ and (BK + 2H+)2+ ions. With pure N2 as the FAIMS carrier gas, more than ten conformers of (BK + H+)+ can be resolved using FAIMS-IMS, as compared to only four conformers resolved using either FAIMS or IMS alone. Interestingly, multiple conformers of (BK + H+)+ were found to have completely different values of FAIMS compensation voltage (CV), while their IMS drift times were essentially the same, which were also proven experimentally to not result from the structural annealing by the collisional heating in the ion funnel. The separations in the FAIMS and IMS dimensions are substantially orthogonal, and the overall resolving power of two-dimensional FAIMS-IMS separation is largely proportional to the product of the separation resolving powers of FAIMS and IMS. Using a gas mixture of N2/He to further improve the resolving power of the FAIMS separation, the total resolving powers of the combined FAIMS and IMS separation were estimated to be about 1020 and 1400 for (BK + H+)+ and (BK + 2H+)2+ ions, respectively, which are significantly higher than the resolving power of any ion mobility-based separation techniques demonstrated so far. The combined FAIMS-IMS can thus be a much more powerful technique to explore the structural diversity of biomolecules.

Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering.

SUMMARYSingle-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition and peptide identification method, transferring identification based on FAIMS filtering (TIFF), to improve the sensitivity and accuracy of label-free scProteomics. TIFF extends the ion accumulation times for peptide ions by filtering out singly charged ions. The peptide identities are assigned by a three-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells, with >1,100 proteins consistently identified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during lipopolysaccharide stimulation and identified time-dependent proteome changes. A record of this paper’s transparent peer review process is included in the supplemental information.

Leveraging Parameter Dependencies in High-Field Asymmetric Waveform Ion-Mobility Spectrometry and Size Exclusion Chromatography for Proteome-wide Cross-Linking Mass Spectrometry.

Ion-mobility spectrometry shows great promise to tackle analytically challenging research questions by adding another separation dimension to liquid chromatography–mass spectrometry. The understanding of how analyte properties influence ion mobility has increased through recent studies, but no clear rationale for the design of customized experimental settings has emerged. Here, we leverage machine learning to deepen our understanding of field asymmetric waveform ion-mobility spectrometry for the analysis of cross-linked peptides. Knowing that predominantly m/z and then the size and charge state of an analyte influence the separation, we found ideal compensation voltages correlating with the size exclusion chromatography fraction number. The effect of this relationship on the analytical depth can be substantial as exploiting it allowed us to almost double unique residue pair detections in a proteome-wide cross-linking experiment. Other applications involving liquid- and gas-phase separation may also benefit from considering such parameter dependencies.

Mapping Protein Structural Evolution upon Unfolding.

In the past, many intensive attempts failed to capture or underestimated the copopulated intermediate conformers from the protein folding/unfolding reaction. We report a promising approach to kinetically trap, resolve, and quantify protein conformers that evolve during unfolding in solution. We conducted acid-induced unfolding of three model proteins (cytochrome c, myoglobin, and lysozyme), and the corresponding reaction aliquots upon decreasing the pH were electrosprayed for high field asymmetric waveform ion mobility spectrometry (FAIMS) measurements. The copopulated conformers were resolved, visualized, and quantified by a two-dimensional mapping of the FAIMS output. Contrary to expectations, all the above proteins appeared metamorphic (multiple-folded conformations) at the physiological pH, and cytochrome c exhibited an unusual "conformational shuttling" before forming the molten globule state. Thus, in contrast to many previous studies, a wide variety of thermodynamically stable intermediate conformers, including compact, molten globule, and partially unfolded forms, was trapped from solution, probing the unfolding mechanism in detail.

Application of zero-phase digital filtering for effective denoising of field asymmetric waveform ion mobility spectrometry signal.

Field asymmetric waveform ion mobility spectrometry (FAIMS) has a great potential to become a portable technology for rapid detection of chemical and biological agents. However, the ion current signals, measured at the exit of the planar FAIMS directly, may contain different types of noises. The peak information in the FAIMS spectrum, such as the compensation voltage (CV) value at the maximum peak intensity (CVP ) and the peak width at half maximum (Wh ), could not be accurately determined under the weak signal condition, which significantly limits the achievable instrument sensitivity, and there are no existing solutions to the problem. This study analyzed the noise type of FAIMS signal in detail, and three different signal processing algorithms, such as median filtering (MF), discrete wavelet transform (DWT), and zero-phase digital filtering (ZDF), were evaluated for their performance in denoising the FAIMS signal. The results show that the standard deviation of CVp obtained from the signal denoised using ZDF algorithm is at least 31.82% smaller as compared to using MF and DWT algorithms. The standard deviation of Wh is at least 45.45% smaller using ZDF algorithm. Moreover, only ZDF algorithm can keep the percentage error for the CV value of the denoised signal to be within 0.50 ± 0.47% of the true CV value, implying the effectiveness of ZDF algorithm in denoising while retaining the integrity of the signal. The ZDF algorithm greatly reduces the analyte peak extraction error and improves the limit of detection in FAIMS measurements.

Predicting ion mobility as a function of the electric field for small ions in light gases

High resolution mobility devices such as Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) and Differential Mobility spectrometers (DMS) use strong electric fields to gas concentration ratios, E/N, to separate ions in the gas phase. While extremely successful, their empirical results show a non-linear, ion-dependent relation between mobility K and E/N that is difficult to characterize. The one-temperature theory Mason-Schamp equation, which is the most widely used ion mobility equation, unfortunately, cannot capture this behavior. When the two-temperature theory is used, it can be shown that the K−E/N behavior can be followed quite closely numerically by equating the effect of increasing the field to an increase in the ion temperature. This is attempted here for small ions in a Helium gas environment showing good agreement over the whole field range. To improve the numerical characterization, the Lennard-Jones (L-J) potentials may be optimized. This is attempted for Carbon, Hydrogen, Oxygen and Nitrogen at different degrees of theory up to the fourth approximation, which is assumed to be exact. The optimization of L-J improves the accuracy yielding errors of about 3% on average. The fact that a constant set of L-J potentials work for the whole range of E/N and for several molecules, also suggests that inelastic collisions can be circumvented in calculations for He. The peculiar K−E/N hump behaviors are studied, and whether mobility increases or decreases with E/N is shown to derive from a competition between relative kinetic energy and the interaction potentials.

Ion Mobility Spectrometry of Superheated Macromolecules at Electric Fields up to 500 Td.

Since its inception in 1980s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) has been implemented at or near ambient gas pressure. We recently developed FAIMS at 15-30 Torr with mass spectrometry and utilized it to analyze amino acids, isomeric peptides, and protein conformers. The separations broadly mirrored those at atmospheric pressure, save for larger proteins that (as predicted) exhibited dipole alignment at ambient but not low pressure. Here we reduce the pressure down to 4.7 Torr, allowing normalized electric fields up to 543 Td-double the maximum in prior FAIMS or IMS studies of polyatomic ions. Despite the collisional heating to ∼1000 °C at the waveform peaks, the proteins of size from ubiquitin to albumin survived intact. The dissociation of macromolecules in FAIMS appears governed by the average ion temperature over the waveform cycle, unlike the isomerization controlled by the peak temperature. The global separation trends in this "superhot" regime extend those at moderately low pressures, with distinct conformers and no alignment as theorized. Although the scaling of the compensation voltage with the field fell below cubic at lower fields, the resolving power increased and the resolution of different proteins or charge states substantially improved.

Advances in high-throughput proteomic analysis

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。

Low-Field Differential Ion Mobility Spectrometry of Dipole-Aligned Macromolecules.

Ion mobility spectrometry (IMS) with mass spectrometry has grown into a powerful approach to simplify complex mixtures, disentangle isomers, and elucidate their geometries. Two established branches are linear IMS based on the absolute mobility K at moderate normalized electric field E/N and field asymmetric waveform IMS (FAIMS) relying on the evolution of K at high E/N causing strong ion heating. Here, we introduce low-field differential IMS (LODIMS), where the field is too weak for significant heating but suffices to lock the permanent macromolecular ion dipoles, producing novel separations based solely on their alignment. The method is demonstrated for a prototypical large protein-albumin. Its oligomers start separating at fields of just 1 kV/cm (4 Td), or ∼5% of those typical for FAIMS. Negligible ion heating at such fields allows preserving fragile species, in particular the noncovalent complexes up to pentamers (332 kDa) destroyed in FAIMS and not detected without it. The separation parameter (compensation field, EC) in this regime scales with the field linearly versus cubically in FAIMS. The dipole moments obtained from threshold fields for alignment and directional cross sections estimated from the slope of said linear EC dependence appear reasonable.

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

Comprehensive LESA Mass Spectrometry Imaging of Intact Proteins by Integration of Cylindrical FAIMS.

The benefits of high field asymmetric waveform ion mobility spectrometry (FAIMS) for mass spectrometry imaging of intact proteins in thin tissue sections have been demonstrated previously. In those works, a planar FAIMS device coupled with a Thermo Elite mass spectrometer was employed. Here, we have evaluated a newly introduced cylindrical FAIMS device (the FAIMS Pro) coupled with a Thermo Fusion Lumos mass spectrometer for liquid extraction surface analysis mass spectrometry imaging of intact proteins in thin tissue sections from rat testes, kidney, and brain. The method makes use of multiple FAIMS compensation values at each location (pixel) of the imaging array. A total of 975 nonredundant protein species were detected in the testes imaging dataset, 981 in the kidney dataset, and 249 in the brain dataset. These numbers represent a 7-fold (brain) and over 10-fold (testes, kidney) improvement on the numbers of proteins previously detected in LESA FAIMS imaging, and a 10-fold to over 20-fold improvement on the numbers detected without FAIMS on this higher performance mass spectrometer, approaching the same order of magnitude as those obtained in top-down proteomics of cell lines. Nevertheless, high throughput identification within the LESA FAIMS imaging workflow remains a challenge.

Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms

Biological functions of many proteins are governed by post-translational modifications (PTMs). In particular, the rich PTM complement in histones controls the gene expression and chromatin structure with major health implications via a combinatoric language. Deciphering that "histone code" is the great challenge for proteomics given an astounding number of possible proteoforms, including isomers with different PTM positions. These must be disentangled on the top- or middle-down level to preserve the key PTM connectivity, which condensed-phase separations failed to achieve. We reported the capability of ion mobility spectrometry (IMS) methods to resolve such isomers for model histone tails. Here, we advance to biological samples, showing middle-down analyses of histones from mouse embryonic stem cells via online chromatography to fractionate proteoforms with distinct PTM sets, differential or field asymmetric waveform IMS (FAIMS) to resolve the isomers, and Orbitrap mass spectrometry with electron transfer dissociation to identify the resolved species.