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Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations. [ABSTRACT FROM AUTHOR]

Amyloid β oligomers (AβOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AβO‐targeting diagnostics and therapeutics, the AβO structures contributing to AD‐associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AβOs stabilized by the bifunctional crosslinker 1,5‐difluoro‐2,4‐dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aβ in a soluble oligomeric conformation. With DFDNB, solutions of Aβ that would otherwise convert to large aggregates instead yield solutions of stable AβOs, predominantly in the 50–300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top–down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.‐injected mice, the DFDNB‐stabilized AβOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AβO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AβOs in structure‐function studies. The structures of amyloid beta oligomers (AβOs) most germane to the pathogenesis of Alzheimer's disease (AD) are ill‐defined. Here, we demonstrate the utility of the crosslinker 1,5‐difluoro‐2,4‐dinitrobenzene (DFDNB) as a tool for AβO stabilization in structure–function studies. We find that DFDNB‐linked AβOs are detergent‐insensitive and remain in a soluble conformation with time and with increased concentration. Importantly, DFDNB stabilization does not alter the ability of AβOs to invoke potent responses than in multiple AD‐relevant pathogenic assays. [ABSTRACT FROM AUTHOR]

Electron transfer dissociation (ETD) has improved the mass spectrometric analysis of proteins and peptides with labile post-translational modifications and larger intact masses. Here, the parameters governing the reaction rate of ETD are examined experimentally. Currently, due to reagent injection and isolation events as well as longer reaction times, ETD spectra require significantly more time to acquire than collision-induced dissociation (CID)) spectra (>100 ms), resulting in a trade-off in the dynamic range of tandem MS analyses when ETD-based methods are compared to CID-based methods. Through fine adjustment of reaction parameters and the selection of reagents with optimal characteristics, we demonstrate a drastic reduction in the time taken per ETD event. In fact, ETD can be performed with optimal efficiency in nearly the same time as CID at low precussor charge state (z = +3) and becomes faster at higher charge state (z > +3). [ABSTRACT FROM AUTHOR]

Top-down proteomics has improved over the past decade despite the significant challenges presented by the analysis of large protein ions. Here, the detection of these high mass species by electrospray-based mass spectrometry (MS) is examined from a theoretical perspective to understand the mass-dependent increases in the number of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quantitative model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of 13C and 15N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, we find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20-fold. [ABSTRACT FROM AUTHOR]

The article offers information on the mass spectrometry for protein complexes. It informs that protein molecules have great importance in biological processes and mass spectrometry has found great utility in the study of protein primary structure. It informs that the noncovalent binding of inhibitors to proteins was shown to be detectable in the gas phase following electrospray ionization.

Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values (R² = 0.6–0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C (R = 0.5) and glycated ApoA-I with higher fasting glucose (R = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single N-acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference (R ∼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes. [ABSTRACT FROM AUTHOR]

Gene therapies based on adeno-associated viruses are an emerging area with high potential to improve human health. Current quality control techniques to assess contaminates and byproducts from the adeno-associated virus (AAV) production pipelines are lacking in robustness and throughput. To address these limitations, we coupled an automated microfluidic device called SampleStream with Orbitrap-based charge detection mass spectrometry (SS-CDMS). We demonstrate that the SS-CDMS workflow performs AAV analysis in under 15 min per sample in a completely autonomous manner. The SS-CDMS workflow enables rapid assessment of key quality control attributes (CQAs), such as of molecular weight and content ratio of AAV formulations with a small sample requirement (<2 × 10 9 capsids) without being limited by sample concentration. Additionally, this work shows the potential for the SS-CDMS workflow to be implemented at various stages of the production pipeline through effective sample clean up from more complex AAV matrices such as cell culture media.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS , a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.

Charge detection mass spectrometry (CDMS) is mainly utilized to determine the mass of intact molecules. Previous applications of CDMS have determined the mass-to-charge ratio and the charge of large polymers, DNA molecules, and native protein complexes, from which corresponding mass values could be assigned. Recent advances have demonstrated that CDMS using an Orbitrap mass analyzer yields the reliable assignment of integer charge states that enables individual ion mass spectrometry (I 2 MS) and spectral output directly into the mass domain. Here I 2 MS analysis was extended to isotopically resolved fragment ions from intact proteoforms for the first time. With a radically different bias for ion readout, I 2 MS identified low-abundance fragment ions containing many hundreds of residues that were undetectable by standard Orbitrap measurements, leading to a doubling in the sequence coverage of triosephosphate isomerase. Thus MS/MS with the detection of individual ions (MS/I 2 MS) provides a far greater ability to detect high mass fragment ions and exhibits strong complementarity to traditional spectral readout in this, its first application to top-down mass spectrometry.

New tools and techniques have dramatically accelerated the field of structural biology over the past several decades. One potent and relatively new technique that is now being utilized by an increasing number of laboratories is the combination of so-called "native" electrospray ionization (ESI) with mass spectrometry (MS) for the characterization of proteins and their noncovalent complexes. However, native ESI-MS produces species at increasingly higher m / z with increasing molecular weight, leading to substantial differences when compared to traditional mass spectrometric approaches using denaturing ESI solutions. Herein, these differences are explored both theoretically and experimentally to understand the role that charge state and isotopic distributions have on signal-to-noise (S/N) as a function of complex molecular weight and how the reduced collisional cross sections of proteins electrosprayed under native solution conditions can lead to improved data quality in image current mass analyzers, such as Orbitrap and FT-ICR. Quantifying ion signal differences under native and denatured conditions revealed enhanced S/N and a more gradual decay in S/N with increasing mass under native conditions. Charge state and isotopic S/N models, supported by experimental results, indicate that analysis of proteins under native conditions at 100 kDa will be 17 times more sensitive than analysis under denatured conditions at the same mass. Higher masses produce even larger sensitivity gains. Furthermore, reduced cross sections under native conditions lead to lower levels of ion decay within an Orbitrap scan event over long transient acquisition times, enabling isotopic resolution of species with molecular weights well in excess of those typically resolved under denatured conditions.

Intact protein mass spectrometry (MS) via electrospray-based methods is often degraded by low-mass contaminants, which can suppress the spectral quality of the analyte of interest via space-charge effects. Consequently, selective removal of contaminants by their mobilities would benefit native MS if achieved without additional hardware and before the mass analyzer regions used for selection, analyte readout, or tandem MS. Here, we use the high-pressure multipole within the source of an Orbitrap Tribrid as the foundation for a coarse ion filter. Using this method, we show complete filtration of 2 mM polyethylene glycol (PEG-1000) during native MS of SILu mAb antibody present at a 200× lower concentration. We also show the generality of the process by rescuing 10 μM tetrameric pyruvate kinase from 2 mM PEG-1000, asserting this voltage rollercoaster filtering (VRF) method for use in native MS as an efficient alternative to conventional purification methods.

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.