I²MS

@article{nokey,

title = {Intact Mass Profiling Reveals Phospho-Proteoforms of the Catenins (85-110 kDa) Regulated by Actomyosin Contractility},

author = {Huang, C.F. and Su, T. and Flozak, A.S. and Gottardi, C.J. and Kelleher, N,L.},

doi = {10.1002/anie.202518593},

year  = {2025},

date = {2025-10-29},

urldate = {2025-10-29},

journal = {Angew Chem Int Ed Engl.},

volume = {Online ahead of print.},

abstract = {A central challenge in top-down proteomics (TDP) is the characterization of large proteoforms (>70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I2MS) for intact mass and fragmentation analysis of β- and α-catenins (85-110 kDa), key components of adherens junctions. Using denatured I2MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on β-catenin (β-cat) and 7 on α-catenin (α-cat), with site-specific changes corresponding to actomyosin contractility. Notably, phosphorylation at α-cat S641 was constitutive, while other sites in the P-linker and actin-binding domains, as well as β-cat S675 and S552, were sensitive to actomyosin perturbation. Application of I2MS for fragment ion detection (I2MS2) also enabled 25%-30% sequence coverage for these exceptionally large proteoforms, compared to <1% using conventional methods for top-down mass spectrometry (MS). Our results are consistent with a "catenin phospho‑code" model, wherein combinatorial phosphorylation patterns reflect and potentially modulate the mechanotransductive environment at cell-cell adhesions. This work establishes top-down I2MS as a viable approach for probing complex post-translational modification (PTM) landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {The Proteoform Landscape of Tau from the Human Brain},

author = {Xu, T. and Su, T. and Soye, B.J.D. and Kandi, S. and Huang, C.F. and Wilkins, J.T. and Castellani, R.J. and Kafader, J.O. and Patrie, S.M. and Vassar, R. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.jproteome.5c00139},

year  = {2025},

date = {2025-06-06},

journal = {Journal of Proteome Research},

volume = {24},

issue = {6},

pages = {2916-2925},

abstract = {Tau is a microtubule-associated protein (MAP) and is critical for maintaining the cytoskeleton of neurons. Tau and its post-translational modifications (PTMs) have been studied for decades, yet the exact composition of intact tau and its truncation products present in the human brain has evaded study at the proteoform level. Here, we show that tau proteoform profiling and exact characterization are possible using immunoprecipitation (IP) and the new approach of individual ion mass spectrometry (I2MS). We provide a first glimpse of the tau proteoform landscape present in the CHAPS-soluble extracts from the temporal cortex of a control subject and a donor with Alzheimer’s disease (AD). Profiling and identification of four isoforms (0N3R, 1N3R, 0N4R, and 1N4R), truncated products (e.g., 72–172 derived from the 0N3R/0N4R isoforms), and intact tau proteoforms harboring PTMs include phosphorylation, methylation, and acetylation. The specific tau proteoform identification typically employs proton transfer charge reduction (PTCR) and electron transfer dissociation (ETD) with spectral readout by individual ion tandem mass spectrometry (I2MS2). A precise understanding of the tau proteoform landscape over the course of neurodegeneration is critical to understand AD pathology vs related dementias. The assay approach reported here will advance AD research, gives a sense of what is technologically possible for new biomarker discovery and will assist the development of therapeutics using the most exact kind of compositional information on tau.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Proteoform profiling of endogenous single cells from rat hippocampus at scale},

author = {Su, P. and Hollas, M.A.R. and Pla, I. and Rubakhin, S. and Butun, F.A. and Greer, J.B. and Early, B.P. and Fellers, R.T. and Caldwell, M.A. and Sweedler, J.V. and Kafader, J.O. and Kelleher, N.L.

},

doi = {https://doi.org/10.1038/s41587-025-02669-x},

year  = {2025},

date = {2025-05-15},

journal = {Nature Biotechnology},

abstract = {We perform intact proteoform profiling of 10,809 endogenous single cells from the rat hippocampus using single-cell proteoform imaging mass spectrometry (scPiMS). scPiMS directly extracts whole proteins and demonstrates high throughput for MS-based single-cell proteomics compared with existing approaches. We develop an informatics workflow dedicated to this datatype and use it to assign neurons, astrocytes or microglia cell types according to their proteoform signatures.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {A Robust and Automated Platform for Charge Detection Mass Spectrometry of Megadalton Biotherapeutics},

author = {Janisse, S.E. and Fellers, R.T. and Raab, S.A. and Goodwin, M.P. and Bowen, K.P. and Lian, Z.J. and Durbin, K.R. and Senko, M.W. and Compton, P.D. and Kafader, J.O. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.analchem.4c06397},

year  = {2025},

date = {2025-03-04},

journal = {Analytical Chemistry},

volume = {97},

issue = {8},

pages = {4549-4555},

abstract = {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 × 109 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers},

author = {Su, P. and McGee, J.P. and Hollas, M.A.R. and Fellers, R.T. and Durbin, K.R. and Greer, J.B. and Early, B.P. and Yip, P.F. and Zabrouskov, V. and Srzentić, K. and Senko, M.W. and Compton, P.D. and Kelleher, N.L. and Kafader, J.O.

},

doi = {doi: 10.1038/s41596-024-01091-y.},

year  = {2025},

date = {2025-01-02},

journal = {Nature Protocols},

volume = {20},

issue = {6},

pages = {1485-1508},

abstract = {Individual ion mass spectrometry (I2MS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, I2MS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. I2MS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution. A mass distribution or 'profile' can be created for any desired sample regardless of composition or heterogeneity. To assist in reducing I2MS analysis to practice, we developed this workflow for data acquisition and subsequent data analysis, which includes (i) protein sample preparation, (ii) attenuation of ion signals to obtain individual ions, (iii) the creation of a charge-calibration curve from standard proteins with known charge states and finally (iv) producing a meaningful mass spectral output from a complex or unknown sample by using the STORIboard software. This protocol is suitable for users with prior experience in mass spectrometry and bioanalytical chemistry. First, the analysis of protein standards in native and denaturing mode is presented, setting the foundation for the analysis of complex mixtures that are intractable via traditional mass spectrometry techniques. Examples of complex mixtures included here demonstrate the relevant analysis of an intact human monoclonal antibody and its intricate glycosylation patterns.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Characterization of the Antibody Response to SARS-CoV-2 Infection in COVID-19 Transplant versus Nontransplant Recipients by Ig-MS},

author = {Des Soye, B.J. and Melani, R.D. and Hollas, M.A.R. and Duan, J. and Patrie, S.M. and Fisher, T.D. and Mattamana, B.B. and Daud, A. and Pinelli, D.F. and Ladner, D.P. and Kelleher, N.L. and Forte, E.

},

doi = {https://doi.org/10.1021/acs.jproteome.4c00285},

year  = {2024},

date = {2024-09-06},

journal = {Journal of Proteome Research},

volume = {23},

issue = {9},

pages = {3944-3957},

abstract = {Solid organ transplant recipients with immunosuppressant regimens to prevent rejection are less able to mount effective immune responses to pathogenic infection. Here, we apply a recently reported mass spectrometry-based serological approach known as Ig-MS to characterize immune responses against infection with SARS-CoV-2 in cohorts of transplant recipients and immunocompetent controls, both at a single early time point following COVID-19 diagnosis as well as over the course of one-month postdiagnosis. We found that the antibody repertoires generated by transplant recipients against SARS-CoV-2 do not differ significantly compared to immunocompetent individuals with regard to repertoire titer, clonality, or glycan composition. Importantly, our study is the first to characterize the evolution of antibody glycan profiles in transplant recipients with COVID-19 disease, presenting evidence that the evolution of glycan composition in these immunocompromised individuals is similar to that in immunocompetent people.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Automated Immunoprecipitation, Sample Preparation, and Individual Ion Mass Spectrometry Platform for Proteoforms},

author = {Des Soye, B.J. and McGee, J.P. and Hollas, M.A.R. and Forte, E. and Fellers, R.T. and Melani, R.D. and Wilkins, J.T. and Compton, P.D. and Kafader, J.O. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.analchem.4c01962},

year  = {2024},

date = {2024-08-14},

journal = {Analytical Chemistry},

volume = {96},

issue = {34},

pages = {13879-13887},

abstract = {Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (I2MS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent I2MS acquisition. This automated workflow has been applied to a cohort of 58 test subjects to determine individualized patient antibody responses to SARS-CoV-2 antigens. Results from a range of serum donors include 37 subject I2MS spectra that contained a positive COVID-19 antibody response and 21 I2MS spectra that contained a negative COVID-19 antibody response. This high-throughput automated I2MS workflow can currently process over 100 samples per week and is general for making immunoprecipitation-MS workflows achieve proteoform resolution.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Single Cell Analysis of Proteoforms},

author = {Su, P. and Hollas, M.A.R. and Butun, F.A. and Kanchustambham, V.L. and Rubakhin, S. and Ramani, N. and Greer, J.B. and Early, B.P. and Fellers, R.T. and Caldwell, M.A. and Sweedler, J.V. and Kafader, J.O. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.jproteome.4c00075},

year  = {2024},

date = {2024-06-07},

journal = {Journal of Proteome Research},

volume = {23},

issue = {6},

pages = {1883-1893},

abstract = {We introduce single cell Proteoform imaging Mass Spectrometry (scPiMS), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single cells dropcast onto glass slides. Sampling and detection of whole proteoforms by individual ion mass spectrometry enable a scalable approach to single cell proteomics. This new scPiMS platform addresses the throughput bottleneck in single cell proteomics and boosts the cell processing rate by several fold while accessing protein composition with higher coverage.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Improved Signal Processing for Mass Shifting Ions in Charge Detection Mass Spectrometry},

author = {Goodwin, M.P. and Grinfeld, D. and Yip, P. and Bowen, K.P. and Kafader, J.O. and Kelleher, N.L. and Senko, M.W.

},

doi = {https://doi.org/10.1021/jasms.3c00435},

year  = {2024},

date = {2024-04-03},

journal = {Journal of the American Society for Mass Spectrometry},

volume = {35},

issue = {4},

pages = {658-662},

abstract = {The quality of data in charge detection mass spectrometry depends on accurate determination of ion charge. While the method of selective temporal overview of resonant ions (STORI) has proven to be highly enabling for determining the charge of ions that survive for variable amounts of time, it assumes that the ion frequency exactly matches the frequency being used in the calculation. Any mismatches result in low charge estimates. To address this, the misSTORI method was developed to correct these discrepancies. This can significantly reduce the charge measurement errors for samples with unstable masses. As an example, the misSTORI approach can eliminate a 5.7% charge determination error for a VP3-only AAV capsid that shifts 25 ppm in mass.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry},

author = {Drown, B.S. and Gupta, R. and McGee, J.P. and Hollas, M.A.R. and Hergenrother, P.J. and Kafader, J.O. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.analchem.3c04758},

year  = {2024},

date = {2024-03-19},

journal = {Analytical Chemistry},

volume = {96},

issue = {11},

pages = {4455-4462},

abstract = {The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a “bird’s eye” view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0–4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry},

author = {Stiving, A.Q. and Foreman, D.J. and VanAernum, Z.L. and Durr, E. and Wang, S. and Vlasak, J. and Galli, J. and Kafader, J.O. and Tsukidate, T. and Li, X. and Schuessler, H.A. and Richardson, D.D.

},

doi = {https://doi.org/10.1021/jasms.3c00309},

year  = {2024},

date = {2024-01-03},

journal = {Journal of the American Society for Mass Spectrometry},

volume = {35},

issue = {1},

pages = {62-73},

abstract = {Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography–multiangle light scattering (SEC-MALS) and field flow fractionation–multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Determining Collisional Cross Sections from Ion Decay with Individual Ion Mass Spectrometry},

author = {Fisher, N.P. and McGee, J.P. and Bowen, K.P. and Goodwin, M. and Senko, M.W. and Kelleher, N.L. and Kafader, J.O.

},

doi = {https://doi.org/10.1021/jasms.3c00340},

year  = {2023},

date = {2023-12-06},

journal = {Journal of the American Society for Mass Spectrometry},

volume = {34},

issue = {12},

pages = {2625-2629},

abstract = {Collision cross section (CCS) measurements determined by ion mobility spectrometry (IMS) provide useful information about gas-phase protein structure that is complementary to mass analysis. Methods for determining CCS without a dedicated IMS system have been developed for Fourier transform mass spectrometry (FT-MS) platforms by measuring the signal decay during detection. Individual ion mass spectrometry (I2MS) provides charge detection and measures ion lifetimes across the length of an FT-MS detection event. By tracking lifetimes for entire ion populations, we demonstrate simultaneous determination of charge, mass, and CCS for proteins and complexes ranging from ∼8 to ∼232 kDa.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Automated imaging and identification of proteoforms directly from ovarian cancer tissue},

author = {McGee, J.P. and Su, P. and Durbin, K.R. and Hollas, M.A.R. and Bateman, N.W. and Maxwell, G.L. and Conrads, T.P. and Fellers, R.T. and Melani, R.D. and Camarillo, J.M. and Kafader, J.O. and Kelleher, N.L.

},

doi = {10.1038/s41467-023-42208-3},

year  = {2023},

date = {2023-10-14},

urldate = {2023-10-14},

journal = {Nature Communications},

volume = {14},

issue = {1},

pages = {6478},

abstract = {The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based multiplexed workflow for top-down tandem MS (MS2) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS2 identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform. AutoPiMS is directly interfaced with multifaceted proteoform imaging MS data modalities for the identification of proteoform signatures in tumor and stromal regions in ovarian cancer biopsies. From a total of ~1000 proteoforms detected by region-of-interest label-free quantitation, we discover 303 differential proteoforms in stroma versus tumor from the same patient. 14 of the top proteoform signatures are corroborated by MSI at 20 micron resolution including the differential localization of methylated forms of CRIP1, indicating the importance of proteoform-enabled spatial biology in ovarian cancer.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Combining Surface-Induced Dissociation and Charge Detection Mass Spectrometry to Reveal the Native Topology of Heterogeneous Protein Complexes},

author = {Du, C. and Cleary, S.P. and Kostelic, M.M. and Jones, B.J. and Kafader, J.O. and Wysocki, V.H.

},

doi = {https://doi.org/10.1021/acs.analchem.3c02185},

year  = {2023},

date = {2023-09-19},

journal = {Analytical Chemistry},

volume = {95},

issue = {37},

pages = {13889-13896},

abstract = {Charge detection mass spectrometry (CDMS) enables the direct mass measurement of heterogeneous samples on the megadalton scale, as the charge state for a single ion is determined simultaneously with the mass-to-charge ratio (m/z). Surface-induced dissociation (SID) is an effective activation method to dissociate non-intertwined, non-covalent protein complexes without extensive gas-phase restructuring, producing various subcomplexes reflective of the native protein topology. Here, we demonstrate that using CDMS after SID on an Orbitrap platform offers subunit connectivity, topology, proteoform information, and relative interfacial strengths of the intact macromolecular assemblies. SID dissects the capsids (∼3.7 MDa) of adeno-associated viruses (AAVs) into trimer-containing fragments (3mer, 6mer, 9mer, 15mer, etc.) that can be detected by the individual ion mass spectrometry (I2MS) approach on Orbitrap instruments. SID coupled to CDMS provides unique structural insights into heterogeneous assemblies that are not readily obtained by traditional MS measurements.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains Using Ig-MS},

author = {Forte, E. and Des Soye, B.J. and Melani, R.D. and Hollas, M.A.R. and Kafader, J.O. and Sha, B.E. and Schneider, J.R. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.jproteome.2c00514},

year  = {2022},

date = {2022-12-02},

journal = {Journal of Proteome Research},

volume = {21},

issue = {12},

pages = {2987-2997},

abstract = {SARS-CoV-2 Omicron (B.1.1.529) and its subvariants are currently the most common variants of concern worldwide, featuring numerous mutations in the spike protein and elsewhere that collectively make Omicron variants more transmissible and more resistant to antibody-mediated neutralization provided by vaccination, previous infections, and monoclonal antibody therapies than their predecessors. We recently reported the creation and characterization of Ig-MS, a new mass spectrometry-based serology platform that can define the repertoire of antibodies against an antigen of interest at single proteoform resolution. Here, we applied Ig-MS to investigate the evolution of plasma antibody repertoires against the receptor-binding domain (RBD) of SARS-CoV-2 in response to the booster shot and natural viral infection. We also assessed the capacity for antibody repertoires generated in response to vaccination and/or infection with the Omicron variant to bind to both Wuhan- and Omicron-RBDs. Our results show that (1) the booster increases antibody titers against both Wuhan- and Omicron- RBDs and elicits an Omicron-specific response and (2) vaccination and infection act synergistically in generating anti-RBD antibody repertoires able to bind both Wuhan- and Omicron-RBDs with variant-specific antibodies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Highly multiplexed, label-free proteoform imaging of tissues by individual ion mass spectrometry},

author = {Su, P. and McGee, J.P. and Durbin, K.R. and Hollas, M.A.R. and Yang, M. and Neumann, E.K. and Allen, J.L. and Drown, B.S. and Butun, F.A. and Greer, J.B. and Early, B.P. and Fellers, R.T. and Spraggins, J.M. and Laskin, J. and Camarillo, J.M. and Kafader, J.O. and Kelleher, N.L.

},

doi = {10.1126/sciadv.abp9929},

year  = {2022},

date = {2022-08-12},

urldate = {2022-08-12},

journal = {Science Advances},

volume = {8},

issue = {32},

abstract = {Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by fourfold compared to reported methods and reveals tissue localization of proteoforms at <80-μm spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization with ion detection using individual ion mass spectrometry. We demonstrate highly multiplexed proteoform imaging of human kidney, annotating 169 of 400 proteoforms of <70 kDa using top-down MS and a database lookup of ~1000 kidney candidate proteoforms, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire},

author = {Melani, R.D. and Des Soye, B.J. and Kafader, J.O. and Forte, E. and Hollas, M. and Blagojevic, V. and Negrão, F. and McGee, J.P. and Drown, B. and Lloyd-Jones, C. and Seckler, H.S. and Camarillo, J.M. and Compton, P.D. and LeDuc, R.D. and Early, B. and Fellers, R.T. and Cho, B.K. and Mattamana, B.B. and Goo, Y.A. and Thomas, P.M. and Ash, M.K. and Bhimalli, P.P. and Al-Harthi, L. and Sha, B.E. and Schneider, J.R. and Kelleher, N.L.},

doi = {https://doi.org/10.1021/acs.jproteome.1c00882},

year  = {2022},

date = {2022-01-07},

journal = {Journal of Proteome Research},

volume = {21},

issue = {1},

pages = {274-288},

abstract = {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 multiparametric 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 applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, 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, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry},

author = {McGee, J.P. and Melani, R.D. and Yip, P.F. and Senko, M.W. and Compton, P.D. and Kafader, J.O. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.analchem.0c03282},

year  = {2021},

date = {2021-02-09},

journal = {Analytical Chemistry},

volume = {93},

issue = {5},

pages = {2723-2727},

abstract = {Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and β-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high m/z by >2.5-fold and >1.6-fold, respectively.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Individual Ion Mass Spectrometry Enhances the Sensitivity and Sequence Coverage of Top-Down Mass Spectrometry},

author = {Kafader, J.O. and Durbin, K.R. and Melani, R.D. and Des Soye, B.J. and Schachner, L.F. and Senko, M.W. and Compton, P.D. and Kelleher, N.L.

},

doi = {https://doi.org/10.1021/acs.jproteome.9b00797},

year  = {2020},

date = {2020-03-06},

urldate = {2020-03-06},

journal = {Journal of Proteome Research},

volume = {19},

issue = {3},

pages = {1346-1350},

abstract = {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 (I2MS) and spectral output directly into the mass domain. Here I2MS analysis was extended to isotopically resolved fragment ions from intact proteoforms for the first time. With a radically different bias for ion readout, I2MS 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/I2MS) 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes},

author = {Kafader, J.O. and Melani, R.D. and Durbin, K.R. and Ikwuagwu, B. and Early, B.P. and Fellers, R.T. and Beu, S.C. and Zabrouskov, V. and Makarov, A.A. and Maze, J.T. and Shinholt, D.L. and Yip, P.F. and Tullman-Ercek, D. and Senko, M.W. and Compton, P.D. and Kelleher, N.L.

},

doi = {10.1038/s41592-020-0764-5},

year  = {2020},

date = {2020-03-02},

urldate = {2020-03-02},

journal = {Nature Methods},

volume = {17},

issue = {4},

pages = {391-394},

abstract = {An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {STORI Plots Enable Accurate Tracking of Individual Ion Signals},

author = {Kafader, J.O. and Beu, S.C. and Early, B.P. and Melani, R.D. and Durbin, K.R. and Zabrouskov, V. and Makarov, A.A. and Maze, J.T. and Shinholt, D.L. and Yip, P.F. and Kelleher, N.L. and Compton, P.D. and Senko, M.W.

},

doi = {https://doi.org/10.1007/s13361-019-02309-0},

year  = {2019},

date = {2019-09-11},

urldate = {2019-09-11},

journal = {Journal of the American Society for Mass Spectrometry},

volume = {30},

issue = {11},

pages = {2200-2203},

abstract = {Charge detection mass spectrometry (CDMS) of low-level signals is currently limited to the analysis of individual ions that generate a persistent signal during the entire observation period. Ions that disintegrate during the observation period produce reduced frequency domain signal amplitudes, which lead to an underestimation of the ion charge state, and thus the ion mass. The charge assignment can only be corrected through an accurate determination of the time of ion disintegration. The traditional mechanisms for temporal signal analysis have severe limitations for temporal resolution, spectral resolution, and signal-to-noise ratios. Selective Temporal Overview of Resonant Ions (STORI) plots provide a new framework to accurately analyze low-level time domain signals of individual ions. STORI plots allow for complete correction of intermittent signals, the differentiation of single and multiple ions at the same frequency, and the association of signals that spontaneously change frequency.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{,

title = {Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins},

author = {Kafader, J.O. and Melani, R.M. and Senko, M.W. and Makarov, A.A. and Kelleher, N.L. and Compton, P.D.},

doi = {10.1021/acs.analchem.8b04519},

year  = {2019},

date = {2019-01-04},

urldate = {2019-01-04},

journal = {Analytical Chemistry},

volume = {91},

issue = {4},

pages = {2776–2783},

abstract = {It is well-known that with Orbitrap-based Fourier-transform-mass-spectrometry (FT-MS) analysis, longer-time-domain signals are needed to better resolve species of interest. Unfortunately, increasing the signal-acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay because of their larger collisional cross-sections, and result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low-ion-count spectra and single-ion processing to reconstruct a conventional m/z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with a resolving power of 677 000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only ∼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}