About

Neil L. Kelleher is a professor involved in the Interdisciplinary Biological Sciences Graduate Program at Northwestern University. His laboratory focuses on top-down proteomics, natural products discovery, and proteoform biology, emphasizing technology development and applications of high-performance mass spectrometry in proteomics and microbial natural products. His research specifically targets the enzymology of natural product biosynthesis, development of Fourier Transform Mass Spectrometry (FTMS) technologies for analyzing intact proteins directly, and translational studies of proteoforms across disease-focused subgroups including cardioproteomics, oncoproteomics, neuroproteomics, and immunoproteomics. Kelleher's pioneering efforts in 'Top Down' proteomics involve fragmenting intact protein ions in the gas phase and creating custom bioinformatics tools to characterize unexpected post-translational modifications (PTMs) in various biological systems such as methane-producing microbes, yeast, and human cancer cells. His work aims to measure chemical modifications to proteins in both hypothesis-driven and discovery modes, constructing, automating, and applying custom mass spectrometry and algorithms to decode the biological 'code' written in protein modifications. His contributions include advancing the understanding of proteoform diversity and modifications, which are key to understanding biological processes and disease mechanisms.

Research topics

• Computer Science
• Biology
• Computational biology
• Genetics
• Cell biology
• Chromatography
• Chemistry
• Bioinformatics
• Nanotechnology
• Organic chemistry
• Telecommunications
• Medicine
• Materials science
• Physics
• Chemical physics
• Immunology
• Anatomy
• Ecology
• Physical chemistry
• Biochemistry
• Data science

Selected publications

• Probing Proteoform Heterogeneity From Single Human Oocytes

  Molecular & Cellular Proteomics · 2026-04-10 · 1 citations

  articleOpen accessSenior author

  Ovarian tissue cryopreservation is a fertility preservation strategy available to prepubertal patients undergoing gonadotoxic treatment who are at risk of developing premature ovarian insufficiency or for those whose treatment cannot be delayed. While cryopreserved tissue can be utilized for ovarian tissue transplantation to restore hormone function and fertility in some of these individuals, those with a high risk of malignant cell reintroduction currently have no options. In vitro maturation (IVM) of isolated immature oocytes from prepubertal patients is not yet successful enough for the clinic, necessitating study of the molecular factors influencing oocyte quality for IVM success. In this study, we characterized intact proteoforms from single human oocytes obtained from ovarian tissue cryopreservation and tissue donation in a donor cohort aged 2 to 33 years old to identify changes in the oocyte proteome across the pubertal transition. Utilizing single-cell proteoform imaging mass spectrometry (scPiMS), which employs a sampling probe to raster over single cells coupled to individual ion mass spectrometry detection, we identified 559 proteins and 769 unique proteoforms across 28 oocytes from eight donors, with an average of 78 unique proteoforms per oocyte. We used scPiMS to selectively sample oocytes and cumulus granulosa cells from single cumulus oocyte complexes to identify proteoforms specific to these cell types. Finally, we determined proteoform landscapes for members of the oocyte-specific subcortical maternal complex (SCMC), KHDC3, and OOEP, and found new proteoforms that differ with donor age. Together, these first-in-class observations provide a foundation for understanding protein-level changes in oocyte biology across puberty to ultimately improve the efficiency of IVM and make fertility restoration options accessible for more patients.

  PublisherDOI

• Targeting Conformational Flexibility of a Reactive Intermediate to Enhance Selectivity of a GABA Aminotransferase Inactivator

  Journal of the American Chemical Society · 2026-02-19 · 1 citations

  articleOpen access

  Currently, mechanism-based inactivators (MBIs) are the only available therapeutic option to target γ-aminobutyric acid aminotransferase (GABA-AT). However, off-target activity against homologous enzymes is a well-recognized challenge for the clinical use of MBIs. For example, CPP-115, an MBI of GABA-AT that completed a Phase I clinical trial, also inactivates ornithine aminotransferase (OAT). Here, we present a comprehensive investigation of an OAT-specific inactivation mechanism for CPP-115 by integrating biochemical experiments, X-ray crystallography, and computational simulations. Unlike in GABA-AT, where CPP-115 forms a noncovalent tight-binding adduct only, a covalent adduct was additionally observed with human OAT (hOAT). Notably, the crystal structures of CPP-115-treated hOAT at different mechanistic stages indicate that the conformational transition of a key intermediate is a prerequisite for the covalent addition pathway. Based on this finding, to selectively reduce the off-target activity, a proof-of-concept molecule that regulates the intermediate conformational flexibility was designed and synthesized. The resulting inactivator achieved greatly enhanced GABA-AT selectivity over OAT and demonstrated therapeutic efficacy in an inflammatory pain animal model. Our strategy in this study, targeting dynamics of a reactive intermediate based on a precise mechanistic understanding, serves as a general design principle for fine-tuning the selectivity of MBIs, particularly for other aminotransferases.

  PublisherOA PDFDOI

• Progressive Protein Network Breakdown in Cirrhosis Progression

  American Journal of Transplantation · 2026-01-01

  articleOpen access
  PublisherOA PDFDOI

• Direct Readout of Multivalent Chromatin Reader-Nucleosome Interactions by Nucleosome Mass Spectrometry

  ACS Central Science · 2026-02-05

  articleOpen accessSenior authorCorresponding

  Histone post-translational modifications (PTMs) often serve as distinct recognition sites for the recruitment of chromatin-associated proteins (CAPs) for epigenome regulation. While CAP:PTM interactions are extensively studied using histone peptides, this cannot represent the regulatory potential of multisite binding on intact nucleosomes. To overcome this limitation, we applied Nucleosome Mass Spectrometry (Nuc-MS), a native Top-Down MS approach that enables the controlled disassembly and proteoform analysis of CAP:nucleosome (CAP:nuc) complexes. As proof of principle, we show the BPTF plant homeodomain (PHD)-bromodomain (BD) native tandem reader binds synergistically to both PTM classes in fully defined ([H3K4me3K9acK14acK18ac]2) nucleosomes. We then extend to explore the engagement of BRD4 (native BD1-BD2), DNMT3A-MPP8 (chimeric PWWP-CD), and Populus trichocarpa Short Half Life (PtSHL) (native bromodomain-adjacent homology (BAH-PHD) tandem readers with endogenous HeLa nucleosomes. In the resulting enrichment profiles, BRD4 favors di- and triacetylated histone H4 proteoforms, whereas DNMT3A-MPP8 and PtSHL recover distinct hypermethylated H3 proteoforms. Of note, PtSHL enriches a potential {H3K4me3K27me3} cis combinatorial that expands the biology of this bivalent signature previously only described in trans. By directly characterizing CAP:nuc complex composition, Nuc-MS informs on the nucleoforms driving binding and thus identifies primary candidates for direct biochemical, structural, and genomic studies.

  PublisherDOI

• Optimizing femtosecond laser interactions towards spatial imaging mass spectrometry

  2026-03-04

  article

  The preservation of biomolecule integrity during femtosecond laser desorption mass spectrometry is governed by nonlinear, wavelength-dependent mechanisms such as multiphoton and avalanche ionization. We evaluated intact signal responses of proteins, and three metabolites using offline laser desorption across wavelengths from the infrared to the deep ultraviolet. Glutamine exhibited strong intact signals in the ultraviolet but not at 515 nm, suggesting electroninduced dissociation, while glucose and proteins retained signals at longer wavelengths but were lost in the deep ultraviolet, likely due to photodissociation from multiphoton ionization. Larger proteins showed reduced signal strength after ultrafast laser desorption, indicating fragmentation driven by increased electron collision cross sections. These findings reveal a pronounced analytespecific dependence on excitation wavelength and underscore the importance of wavelength selection for optimizing molecular integrity in ultrafast laser desorption mass spectrometry.

  PublisherDOI

• PSLite Online: A Portable, Modern Web Application to Analyze Top‐Down Mass Spectrometry Fragmentation Data

  Journal of Mass Spectrometry · 2026-02-12

  articleOpen access

  PSLite Online is a modern, web-based application designed to facilitate the analysis of top-down mass spectrometry fragmentation data, particularly for targeted proteoform studies. As a successor to the Windows-only ProSight Lite, PSLite Online retains and expands upon its core functionalities while offering platform independence and enhanced usability through a responsive, browser-based interface. Users can load or manually define proteoforms, apply a wide range of modifications, and visualize fragmentation coverage in real time. Key new features include ProForma integration, correction for common mass deconvolution errors, SVG export, and cloud-based session sharing via persistent URLs. The application leverages modern web technologies and component libraries to deliver a lightweight, installable progressive web app (PWA) experience. Here, we show that PSLite Online can be used for the characterization of antibody subunits with glycan modifications. This solution broadens accessibility for proteomics researchers by eliminating installation barriers and enabling seamless collaboration across devices and platforms. PSLite Online is freely available at https://pslite.proteinaceous.net.

  PublisherOA PDFDOI

• Top-Down Proteomics in the Assessment of Kidney Donor Quality: a novel approach to increased organ utilization

  medRxiv · 2026-02-04

  articleOpen accessCorresponding

  Abstract Kidney transplantation faces a critical paradox: while thousands await organs, approximately 30% of potential deceased donor kidneys are discarded for various reasons, including subjective assessments due to the lack of an objective molecular biomarker of preservation quality. Here, we applied novel “top-down” proteoform imaging mass spectrometry across living donor (LD), deceased donor (brain death or cardiac death), and discarded human kidneys to quantify proteoforms correlating with post-transplant kidney function. This approach preserves post-translational modifications and splice variants, revealing molecular tissue variability beyond protein presence. LD kidneys displayed robust metabolic signatures, including L-xylulose reductase and cytochrome oxidase subunits, whereas deceased donor and discarded organs showed elevated cellular stress markers such as alpha-B-crystallin and peroxiredoxin 1. Post-transplant blood proteoform analysis validated tissue findings, demonstrating persistent cellular stress and immune activation in deceased donor recipients compared with physiologic wound healing in LD recipients. Consistent with these molecular predictions, serum creatinine levels were highest in DCD, intermediate in DBD, and lowest in LD recipients. The intersection of tissue proteoform signatures across all marginal tissues identified four proteoforms consistently elevated in deceased and discarded kidneys: ACTG1, acetylated CRYAB, PARK7, and S100A4. Collectively, these proteoforms capture key molecular indicators of graft quality, reflecting oxidative stress, cellular injury, and immune activation pathways. As such, they represent promising point-of-care (POC) biomarker candidates for objective kidney classification, potentially improving donor kidney utilization. Translational statement Current methods for evaluating donor kidney quality rely on subjective assessments, contributing to the discard of approximately 30% of potentially viable organs. This study demonstrates that “top-down” proteomics can objectively identify molecular signatures distinguishing high-quality from marginal donor kidneys. Top-down proteomics analyzes intact proteins with their post-translational modifications or cleavage products, termed proteoforms to provide mechanistic insights into graft quality. We identified four proteoforms (ACTG1, acetylated CRYAB, PARK7, and S100A4) to be consistently elevated in deceased and discarded kidneys, reflecting oxidative stress, cellular injury, and immune activation. These molecular markers correlated with post-transplant kidney outcome, as measured by serum creatinine levels and recipient blood proteoforms. As a next step, validation in larger cohorts could establish these proteoforms as point-of-care biomarkers for real-time donor kidney assessment during procurement. This objective molecular stratification could reduce unnecessary organ discards and improve transplant outcomes by matching organ quality with recipient risk profiles.

  PublisherOA PDFDOI

• Proteoform medicine: characterizing and targeting protein forms in human disease

  Nature Reviews Genetics · 2026-01-05 · 7 citations

  articleOpen access
  PublisherOA PDFDOI

• Automated Enzymatic Deglycosylation of Monoclonal Antibody with the SampleStream Platform

  Journal of the American Society for Mass Spectrometry · 2025-12-08

  articleOpen accessSenior authorCorresponding

  Engineered monoclonal antibodies have proven themselves as invaluable biotherapeutics used in clinics worldwide. In discovery, development, and production, it is essential that they are accurately validated to ensure their homogeneity, consistency, safety, and effectiveness, which are commonly conducted via mass spectrometric analysis. However, validation processes can be manual, time-consuming, and costly. One hindrance in the analytical workflow is the relatively long time required to deglycosylate native mAbs with PNGase F on the benchtop, which is usually done to reduce the spectral complexity, increase the ionization efficiency, and facilitate the identification of glycosylation sites. To circumvent this obstacle, a workflow on the SampleStream Platform was developed and optimized to automate in-channel PNGase F-mediated deglycosylation of native monoclonal antibody at unprecedented speed, accomplishing 86% deglycosylation in 3 min. The presented workflow offers a promising strategy to reduce discovery and development costs and streamline the characterization of antibody-based biopharmaceuticals.

  PublisherOA PDFDOI

• A needed nomenclature for nucleosomes

  Molecular Cell · 2025-10-01 · 9 citations

  reviewOpen access
  PublisherOA PDFDOI

Recent grants

• Molecular Profiling & Characterization

  NIH · $18.2M · 2004–2029

• NIH Grant R01GM067193

  NIH · $4.0M · 2018

• Cancer Research Training and EducationCoordination

  NIH · $51.1M · 1997–2030

• Renewable and Specific Affinity Reagents for Mapping Proteoforms in Human Tissues

  NIH · $384k · 2019–2022

• NIH Grant R01GM067725

  NIH · $3.0M · 2016

Frequent coauthors

• Paul M. Thomas

  Radiometrics (United States)

  159 shared

• Jeannie M. Camarillo

  Northwestern University

  127 shared

• Christopher T. Walsh

  Met Office

  110 shared

• Ryan T. Fellers

  Northwestern University

  82 shared

• Philip D. Compton

  Bristol-Myers Squibb (United States)

  72 shared

• Rafael D. Melani

  Northwestern University

  72 shared

• Amanda Saratsis

  64 shared

• Mandana Behbahani

  Montefiore Medical Center

  58 shared