CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH†.

Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S)x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S)x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S)x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S)x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S)x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound end-to-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.

Modified Ion Source for the Improved Collisional Activation of Protein Complexes.

The analysis of large molecules is challenging, as they often have salts and adducts retained through the electrospray process, which increase the observed mass and compromise the achievable mass resolution. Mild collisional activation has been shown to be very effective for the removal of adducts and increases both measurement accuracy and mass resolution of large (>100 kDa) protein complexes. Collisionally activated protein ions are more completely desolvated due to the increased number of collisions when trapped following activation. A short square quadrupole maintained at 300 mTorr by a mechanical pump was added between the ion funnel and transmission quadrupole. This configuration and operation effectively removed adducts from the 800 kDa tetradecamer GroEL as well as fragmented smaller protein complexes like C-reactive protein. Due to the gas high pressure, ions of low size-to-charge ratio, such as those in charge reducing buffers, had low ejection efficiency. We show that segmenting the quadrupole rods greatly improves signal intensity for charge reduced GroEL D398A mutant compared to nonsegmented rods when operating at high pressure.

Automated and Miniaturized Pico-Liter Metabolite Extraction System for Single-Cell Mass Spectrometry.

OBJECTIVE: Mass spectrometry has become the method of choice for single cell analysis due to its high sensitivity of detection and capability in analyzing a large number of metabolites simultaneously. For a long time, an automated and miniaturized system capable of extracting cellular contents from single cells at the pico-liter level for pico-ESI analysis has been lacking. METHODS: This paper presents a first-of-its-kind automated and miniaturized pico-liter extraction system for single-cell MS. The key modules, including imaging, bus controller, and fluidic driving are customized to achieve satisfactory performance at affordable costs, resulting in a miniaturized system movable on a trolley and connectable with the MS. To enable automation, a single cell trapping device, new image-based one-pixel accuracy positioning methods for cells and micropipette, and a surface-tension-based 1-pL accuracy volume control scheme are developed. RESULTS: The system is able to control the solvent loading at 1.97 ± 0.05 nL, solvent dispensing at 14-15 pL, and solvent evaporation at 689±48 pL. MS experiments demonstrate a throughput of 20 cells/h. CONCLUSION: The system has achieved better performance in consistency (∼21%), sensitivity (∼28%), and success rate (up to 40%) than manual operation. SIGNIFICANCE: This automated and miniaturized system lays a solid basis for applying pico-ESI MS analysis in the automated and high-throughput single cell MS analysis, such as single-cell metabolomics and lipidomics.

Building Australia's first mass spectrometer: Recognising the achievements of J. Roger Bird.

Following the birth of the field of mass spectrometry at the end of World War I, it was several decades before the first commercial mass spectrometers became available. In the interim, many physicists interested in the nature of matter, and their application to studies in nuclear physics, constructed their own. A young physics postgraduate student, John Roger Bird, was the first to do so in Australia. This article describes his efforts and achievements, featuring technical blueprints, photographs of the instruments and early data, in long overdue recognition of Bird's work at the University of Melbourne.

Recent technological developments for native mass spectrometry.

Native mass spectrometry (MS), the analysis of proteins and protein complexes from solutions that stabilize native solution structures, is a rapidly expanding area. There is strong evidence supporting the retention of proteins' native folds in the absence of solvent under the experimental timescales of MS experiments. Therefore, instrumentation has been developed to use gas-phase native-like protein ions to exploit the speed, sensitivity, and selectivity of mass spectrometry approaches to solve emerging problems in structural biology. This article reviews some of the recent advances and applications in gas-phase instrumentation for structural proteomics.

Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Development and validation of an LC-MS/MS method for pharmacokinetic study of lobetyolin in rats

Abstract A simple and selective liquid chromatography tandem with mass spectrometry (LC-MS/ MS) method for quantification of lobetyolin in rat plasma was developed and validated. Chromatographic separation was achieved on a Thermo ODS C18 reversed-phase column using 0.1% aqueous formic acid-methanol (50:50, v/v) in an isocratic elution mode at a flow rate of 0.4 mL.min-1. LC/MS performance was done in a positive ion ESI mode and the MS/MS transitions were monitored at m/z 419.3 [M+Na]+ → m/z 203.1 for lobetyolin and m/z 394.9 [M+Na]+ → m/z 231.9 for IS, respectively. The assay exhibited a linear dynamic range over 1.0-500 ng.mL-1 for lobetyolin in plasma. Both the precision (%RSD) and accuracy (RE%) were within acceptable criteria (<15%). Recoveries ranged from 87.0% to 95.6%, and the matrix effects were from 91.0% to 101.3%. After oral administration, the peak plasma concentration of lobetyolin was obtained as 60.1 ng.mL-1 at 1.0 h. The proposed LC-MS/MS method could be applied to a pharmacokinetic study employing 66 samples from 6 Wistar rats

VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function.

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.

Regulated and Non-Regulated Mycotoxin Detection in Cereal Matrices Using an Ultra-High-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UHPLC-HRMS) Method.

Cereals represent a widely consumed food commodity that might be contaminated by mycotoxins, resulting not only in potential consumer health risks upon dietary exposure but also significant financial losses due to contaminated batch disposal. Thus, continuous improvement of the performance characteristics of methods to enable an effective monitoring of such contaminants in food supply is highly needed. In this study, an ultra-high-performance liquid chromatography coupled to a hybrid quadrupole orbitrap mass analyzer (UHPLC-q-Orbitrap MS) method was optimized and validated in wheat, maize and rye flour matrices. Nineteen analytes were monitored, including both regulated mycotoxins, e.g., ochratoxin A (OTA) or deoxynivalenol (DON), and non-regulated mycotoxins, such as ergot alkaloids (EAs), which are analytes that are expected to be regulated soon in the EU. Low limits of quantification (LOQ) at the part per trillion level were achieved as well as wide linear ranges (four orders of magnitude) and recovery rates within the 68-104% range. Overall, the developed method attained fit-for-purpose results and it highlights the applicability of high-resolution mass spectrometry (HRMS) detection in mycotoxin food analysis.

Source apportionment and quantification of liquid and headspace leaks from closed system drug-transfer devices via Selected Ion Flow Tube Mass Spectrometry (SIFT-MS).

A system to differentiate and quantify liquid and headspace vapor leaks from closed system drug-transfer devices (CSTDs) is presented. CSTDs are designed to reduce or eliminate hazardous drug (HD) exposure risk when compounding and administering HDs. CSTDs may leak liquid, headspace, or a mixture of the two. The amount of HD contained in liquid and headspace leaks may be substantially different. Use of a test solution containing two VOCs with differences in ratios of VOC concentrations in the headspace and liquid enables source apportionment of leaked material. SIFT-MS was used to detect VOCs from liquid and headspace leaks in the vapor phase. Included in this report is a novel method to determine the origin and magnitude of leaks from CSTDs. A limit of leak detection of 24 µL of headspace vapor and 0.14 µL of test liquid were found using Selected Ion Flow Tube Mass Spectrometry (SIFT-MS).

Assessment of Breathomics Testing Using High-Pressure Photon Ionization Time-of-Flight Mass Spectrometry to Detect Esophageal Cancer.

Importance: A triage test is needed to increase the detection rate for esophageal cancer. Objective: To investigate whether breathomics can detect esophageal cancer among patients without a previous diagnosis of cancer using high-pressure photon ionization time-of-flight mass spectrometry (HPPI-TOFMS). Design, Setting, and Participants: This diagnostic study included participants who planned to receive an upper endoscopy or surgery of the esophagus at a single center in China. Exhaled breath was collected with a self-designed collector and air bags before participants underwent these procedures. Sample collection and analyses were performed by trained researchers following a standardized protocol. Participants were randomly divided into a discovery data set and a validation data set. Data were collected from December 2020 to March 2021. Exposures: Breath samples were analyzed by HPPI-TOFMS, and the support vector machine algorithm was used to construct a detection model. Main Outcomes and Measures: The accuracy of breathomics was measured by the sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve. Results: Exhaled breath samples were obtained from 675 patients (216 [32%] with esophageal cancer; 459 [68%] with noncancer diseases). Of all patients, 206 (31%) were women, and the mean (SD) age was 64.0 (11.9) years. In the validation data set, esophageal cancer was detected with an accuracy of 93.33%, sensitivity of 97.83%, specificity of 83.72%, positive predictive value of 94.74%, negative predictive value of 92.78%, and area under the receiver operating characteristic curve of 0.89. Notably, for 16 patients with high-grade intraepithelial neoplasia, 12 (75%) were predicted to have esophageal cancer. Conclusions and Relevance: In this diagnostic study, testing breathomics using HPPI-TOFMS was feasible for esophageal cancer detection and totally noninvasive, which could help to improve the diagnosis of esophageal cancer.

Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool: A cross-sectional proof-of-concept study.

Clostridioides difficile infection (CDI) is an important infectious cause of antibiotic-associated diarrhea, with significant morbidity and mortality. Current diagnostic algorithms are based on identifying toxin by enzyme immunoassay (EIA) and toxin gene by real-time polymerase chain reaction (PCR) in patients with diarrhea. EIA's sensitivity is poor, and PCR, although highly sensitive and specific, cannot differentiate infection from colonization. An ideal test that incorporates microbial factors, host factors, and host-microbe interaction might characterize true infection, and assess prognosis and recurrence. The study of volatile organic compounds (VOCs) has the potential to be an ideal diagnostic test. The presence of VOCs accounts for the characteristic odor of stool in CDI but their presence in breath and plasma has not been studied yet. A cross-sectional proof-of-concept study analyzing VOCs using selected ion flow tube mass spectrometry (SIFT-MS) was done on breath, stool, and plasma of patients with clinical features and positive PCR for CDI (cases) and compared with patients with clinical features but a negative PCR (control). Our results showed that VOC patterns in breath, stool, and plasma, had good accuracy [area under the receiver operating characteristic curve (ROC) 93%, 86%, and 91%, respectively] for identifying patients with CDI.

Requirements and attributes of nano-resonator mass spectrometry for the analysis of intact viral particles.

When studying viruses, the most prevalent aspects that come to mind are their structural and functional features, but this leaves in the shadows a quite universal characteristic: their mass. Even if approximations can be derived from size and density measurements, the multi MDa to GDa mass range, featuring a majority of viruses, has so far remained largely unexplored. Recently, nano-electromechanical resonator-based mass spectrometry (NEMS-MS) has demonstrated the ability to measure the mass of intact DNA filled viral capsids in excess of 100 MDa. However, multiple factors have to be taken in consideration when performing NEMS-MS measurements. In this article, phenomena influencing NEMS-MS mass estimates are listed and discussed, including some particle's extraneous physical properties (size, aspect ratio, stiffness), and the influence of frequency noise and device fabrication defects. These factors being accounted for, we could begin to notice subtler effects linked with (e.g.) particle desolvation as a function of operating parameters. Graphical abstract.

Surface-assisted Laser Desorption/ionization Mass Spectrometry Analysis of the Glycolipid Biosurfactants, Mannosylerythritol Lipids, Using an Ionization-assisting Substrate.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for the screening of glycolipid-type biosurfactants (BSs) from a crude extract of microbial products. However, it is unsuitable for the detection of lower molecular weight products because the observed ions are overlapped with matrix-derived ions at lower mass range. In this study, we applied a "matrix-free" surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) analysis using a through-hole alumina membrane as an ionization-assisting substrate. Using this method, we could detect a variety of lower molecular weight products in an extract of a glycolipid BS producer with good sensitivity. In addition, the culture solution could be analyzed directly by this method.

Expanding Molecular Coverage in Mass Spectrometry Imaging of Microbial Systems Using Metal-Assisted Laser Desorption/Ionization.

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (∼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.

Confined DART-MS for Rapid Chemical Analysis of Electronic Cigarette Aerosols and Spiked Drugs.

A confined direct analysis in a real time mass spectrometry (DART-MS) system and method were developed for coupling directly with commercial electronic cigarettes for rapid analysis without sample preparation. The system consisted of a confining heated glass T-junction, DART ionization source, and Vapur interface to assist aerodynamic transport. Suction generated by positioning the electronic cigarette at the junction inlet allowed for direct chemical analysis of aerosolized electronic liquids from both automatic devices powered by drag and manual button-operated devices, which is unachievable with traditional DART-MS. Parametric analyses for the system investigated Vapur suction flow rate, junction heating, puff duration, and coil power levels. Using this method, rapid chemical analyses of electronic cigarette aerosols from electronic liquids, spiked illicit drugs, and polymeric or plasticizer contaminants were performed in <30 s. The confined DART-MS method provides a streamlined tool for rapid screening of illicit and hazardous chemical profiles emitting from electronic cigarettes.

On-surface autosampling for liquid chromatography-mass spectrometry.

An on-surface multi-purpose autosampler was built for liquid chromatography-mass spectrometry (LC-MS) based on the autoTLC-MS interface, taking advantage of open-source hard- and software developments as well as 3D printing. Termed autoTLC-LC-MS system, it is introduced for orthogonal hyphenation of normal phase high-performance thin-layer chromatography with reversed phase high-performance LC (HPLC) and high-resolution MS (HRMS). For verification of its functionality, a multi-class antibiotic mixture was applied as a calibration band pattern on an adsorbent layer and detected by the Bacillus subtilis bioassay. This effect-image was uploaded as a template in the updated TLC-MS_manager software. The clicked-on antibiotic zones were sequentially eluted without intervention from the planar counterpart (without bioassay) via a monolithic HPLC column into the HRMS system. For elution of antibiotics of 7 structural classes at 5 different calibration levels, the new on-surface autosampler achieved intra-day precisions of 2.1-14.1%, while inter-day precisions ranged 2.5-16.1% (all n = 3). The new hyphenation offers potential for planar sample clean-up prior to HPLC, concentration of liquid samples, increase of peak capacity and proof of peak purity or isomers. The integrated autoTLC-LC-MS system enabled high sample throughput, efficiency and reproducibility for the first time through fully automated TLC-LC-MS sequence operation. Its contact-closure signal functionality, versatile 3D printed planar sample holder and open-source software made it readily adjustable for new analytical tasks. Undoubtedly, any planar material can be investigated for leachables, such as textiles, foils, papers and other packagings, as well as planar biological samples for ingredients.

Applications of mass spectrometry imaging in virus research.

Mass spectrometry imaging (MSI) is a label-free molecular imaging technique allowing an untargeted detection of a broad range of biomolecules and xenobiotics. MSI enables imaging of the spatial distribution of proteins, peptides, lipids and metabolites from a wide range of samples. To date, this technique is commonly applied to tissue sections in cancer diagnostics and biomarker development, but also molecular histology in general. Advances in the methodology and bioinformatics improved the resolution of MS images below the single cell level and increased the flexibility of the workflow. However, MSI-based research in virology is just starting to gain momentum and its full potential has not been exploited yet. In this review, we discuss the main applications of MSI in virology. We review important aspects of matrix-assisted laser desorption/ionization (MALDI) MSI, the most widely used MSI technique in virology. In addition, we summarize relevant literature on MSI studies that aim to unravel virus-host interactions and virus pathogenesis, to elucidate antiviral drug kinetics and to improve current viral disease diagnostics. Collectively, these studies strongly improve our general understanding of virus-induced changes in the proteome, metabolome and metabolite distribution in host tissues of humans, animals and plants upon infection. Furthermore, latest MSI research provided important insights into the drug distribution and distribution kinetics, especially in antiretroviral research. Finally, MSI-based investigations of oncogenic viruses greatly increased our knowledge on tumor mass signatures and facilitated the identification of cancer biomarkers.

Dissociation Strategies to Maximize Coverage of α-Helical Domains in Top-Down Mass Spectrometry of Integral Membrane Proteins.

Transmembrane α-helical domains of membrane proteins tend to remain structured in the gas phase, presenting a challenge for efficient electron capture/transfer dissociation during top-down dissociation mass spectrometry (MS) experiments. In this study, we compare results from different dissociation modes on a modern Orbitrap platform applied to a model integral membrane protein containing two transmembrane helices, the c-subunit of the Fo domain of the chloroplast ATP synthase. Using commercially available options, we compare collisionally activated dissociation (CAD) with the related variant higher-energy collisional dissociation (HCD) and with electron transfer dissociation (ETD). HCD performed better than CAD and ETD. A combined method utilizing both ETD and HCD (EThcD) demonstrates significant synergy over HCD or ETD alone, representing a robust option analogous to activated ion electron capture dissociation, whereby an infrared laser was used to heat the protein ion alongside electron bombardment. Ultraviolet photodissociation at 213 nm displays at least three backbone dissociation mechanisms and covered nearly 100% of backbone bonds, suggesting significant potential for this technique.

New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry.

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.