About

Miklos Guttman is an Associate Professor in the Department of Medicinal Chemistry at the University of Washington. His research focuses on antibody-antigen recognition, a critical biological process underlying immune responses and the mechanisms of biotherapeutics and vaccines. His lab utilizes emerging biophysical and structural tools such as structural mass spectrometry to characterize interactions of intact antibody-antigen complexes in their native solution state. This work aims to advance the development of biotherapeutic approaches against infectious diseases and cancer, with current investigations into antibody-mediated neutralization of staphylococcal enterotoxins and cancer recognition by immunoglobulin μ (IgM). Additionally, Guttman's lab explores the role of glycosylation in biology, developing and implementing novel mass spectrometry-based methods to obtain higher levels of structural information for biologically relevant glycans and oligosaccharides, addressing the analytical challenges posed by the structural complexity of carbohydrates.

Research topics

• Biology
• Immunology
• Virology
• Chemistry
• Biochemistry
• Nanotechnology
• Medicine
• Chromatography
• Computational biology
• Biochemical engineering
• Cell biology
• Biophysics
• Genetics

Selected publications

• Activation mechanism of small heat shock protein HSPB5 revealed by disease-associated mutants

  Proceedings of the National Academy of Sciences · 2025-05-16 · 9 citations

  articleOpen access

  Found from bacteria to humans, small heat shock proteins (sHSPs) are the least understood protein chaperones. HSPB5 (or αB-crystallin) is among the most widely expressed of the 10 human sHSPs, including in muscle, brain, and eye lens where it is constitutively present at high levels. A high content of disorder in HSPB5 has stymied efforts to uncover how its structure gives rise to function. To uncover its mechanisms of action, we compared human HSPB5 and two disease-associated mutants, R120G and D109H. Expecting to learn how the mutations lead to loss of function, we found instead that the mutants are constitutively activated chaperones while wild-type HSPB5 can transition reversibly between nonactivated (low activity) and activated (high activity) states in response to changing conditions. Techniques that provide information regarding interactions and accessibility of disordered regions revealed that the disordered N-terminal regions (NTR) that are required for chaperone activity exist in a complicated interaction network within HSPB5 oligomers and are sequestered from solvent in nonactivated states. Either mutation or an activating pH change causes rearrangements in the network that expose parts of the NTR, making them more available to bind an aggregating client. Although beneficial in the short-term, failure of the mutants to adopt a state with lower activity and lower NTR accessibility leads to increased coaggregation propensity and, presumably, early cataract. The results support a model where chaperone activity and solubility are modulated through the quasi-ordered NTR and its multiple competing interactions.

  PublisherDOI

• Tau4RD fibril polymorphism is imprinted during early aggregation

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-14

  preprintOpen access

  Abstract Microtubule-associated protein tau forms characteristic fibrillar species in many neurodegenerative diseases. Neurofibrillary tangles, tau deposits observed in Alzheimer’s disease (AD), contain a mixture of amyloid-type polymorphic fibrils called paired helical filaments (PHFs) and straight filaments. The formation of heterogenous fibril populations is observed in other diseases and when tau aggregation is induced in vitro with polyanionic species. This suggests that tau’s structural transition from a conformational ensemble to various amyloid morphologies is a controlled and, therefore, controllable process. Despite many years of work toward describing aggregation intermediates that could address open questions such as whether fibril polymorphism is imprinted at the start of aggregation or arises due to conformational conversions, our understanding of amyloid structure remains predominantly based on observations of mature fibrils. It is unclear whether these processes are mutually exclusive and to what extent we can bias intermediate conformations toward less toxic states. Here to address the challenge of studying aggregation intermediates and tau’s structural conversion, we apply pulsed hydrogen-deuterium exchange with mass spectrometry (pulsed HDX-MS), which revealed differences in the subpopulations formed by tau4RD (a truncated tau construct) within seconds of initiating aggregation with polyphosphate and within hours of heparin-induction. This work begins to address the gap in knowledge regarding whether amyloid polymorphism is directly imprinted during nucleation or results from structural rearrangement during later stages of aggregation.

  PublisherOA PDFDOI

• Comprehensive Internal Standards for Hydrogen/Deuterium Exchange Mass Spectrometry

  ChemRxiv · 2025-04-28 · 1 citations

  preprintOpen accessSenior author

  Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a widespread tool for studying protein structure and dynamics, but data comparability across different experimental conditions, instrument platforms, and laboratories remains challenging. Here, we design, synthesize, and validate a novel set of imidazolium-based internal exchange reporters (IERs) that readily integrate with HDX-MS to provide a direct measure of exchange conditions. The set of eight IERs are designed to have a single discrete exchangeable site to sample conditions over exceptionally broad timescales, from milliseconds to days while also exhibiting desirable chromatographic behavior. We demonstrated the utility of these IERs and the associated computational tools through a series of comparative HDX-MS studies involving well studied proteins where reaction conditions like pH and deuterium content were offset. Through unambiguous referencing of exchange reaction conditions, this novel set of internal standards enables rigorous cross-experiment comparisons and will improve HDX-MS derived models of protein dynamics

  PublisherOA PDFDOI

• Rigorous Analysis of Multimodal HDX-MS Spectra

  Journal of the American Society for Mass Spectrometry · 2025-01-21 · 20 citations

  articleSenior authorCorresponding

  An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data.

  PublisherDOI

• Large-scale discovery, analysis and design of protein energy landscapes

  Nature · 2025-03-25 · 11 citations

  preprintOpen access

  All folded proteins continuously fluctuate between their low-energy native structures and higher energy conformations that can be partially or fully unfolded. These rare states influence protein function, interactions, aggregation, and immunogenicity, yet they remain far less understood than protein native states. Although native protein structures are now often predictable with impressive accuracy, conformational fluctuations and their energies remain largely invisible and unpredictable, and experimental challenges have prevented large-scale measurements that could improve machine learning and physics-based modeling. Here, we introduce a multiplexed experimental approach to analyze the energies of conformational fluctuations for hundreds of protein domains in parallel using intact protein hydrogen-deuterium exchange mass spectrometry. We analyzed 5,778 domains 28-64 amino acids in length, revealing hidden variation in conformational fluctuations even between sequences sharing the same fold and global folding stability. Site-resolved hydrogen exchange NMR analysis of 13 domains showed that these fluctuations often involve entire secondary structural elements with lower stability than the overall fold. Computational modeling of our domains identified structural features that correlated with the experimentally observed fluctuations, enabling us to design mutations that stabilized low-stability structural segments. Our dataset enables new machine learning-based analysis of protein energy landscapes, and our experimental approach promises to reveal these landscapes at unprecedented scale.

  PublisherDOI

• Germline encoded residues dominate the interaction of a human monoclonal antibody with decorin binding protein A of Borrelia burgdorferi

  Frontiers in Immunology · 2025-07-07 · 5 citations

  articleOpen access

  During the course of Lyme disease, humans mount a robust and sustained antibody response against dozens of Borrelia burgdorferi outer surface lipoproteins. Identifying which antibodies are associated with spirochete clearance and disease resolution is of paramount importance in therapeutic development. In this study, we describe the isolation and structural characterization of a human monoclonal antibody (MAb) against decorin binding protein A (DbpA), one of the most immunogenic of B. burgdorferi ’s outer surface proteins. High-resolution epitope mapping by HDX-MS and X-ray crystallography revealed that F945 associates with a lateral face of DbpA in a side-on orientation without obstructing resides associated with DbpA’s ability to bind components of the extracellular matrix. The structure of the DbpA-F945 Fab complex revealed an outsized role for variable light chain (V L ) germline encoded residues in mediating DbpA interactions. In fact, the majority of the critical contacts between F945 and DbpA involved V k 1–33 germline encoded residues, suggesting that certain human B cell receptors (BCR) may be preconfigured to recognize DbpA and therefore have a lower threshold for B cell activation and clonal development. Passive administration of F945 IgG was not sufficient to protect against B. burgdorferi in a mouse model of needle infection, although these experiments do not rule out a role for F945 in influencing B. burgdorferi tissue tropism and/or retention within specific niches. Nonetheless, it is tempting to speculate that F945 represents a class of DbpA antibodies with value in Lyme disease diagnostics, but that may not contribute to B. burgdorferi clearance or disease resolution in humans.

  PublisherOA PDFDOI

• High-Throughput Determination of Exchange Rates of Unmodified and PTM-Containing Peptides Using HX-MS

  Molecular & Cellular Proteomics · 2025-01-07 · 5 citations

  articleOpen access

  Despite the widespread use of MS for hydrogen/deuterium exchange measurements, no systematic, large-scale study has been conducted to compare the observed exchange rates in protein-derived, unstructured peptides measured by MS to the predicted exchange rates calculated from NMR-derived values and how neighboring residues and post-translational modifications influence those exchange rates. In this study, we sought to test the accuracy of predicted values by performing hydrogen exchange measurements on whole cell digests to generate an unbiased dataset of 563 unique peptides derived from naturally-occurring protein sequences. A remarkable 97% of observed exchange rates of peptides are within two-fold of predicted values. Using fully deuterated controls, we found that for approximately 50% of the peptides, the amino acid sequence and, consequently, the intrinsic exchange rate, are the primary contributors to back exchange. A meta-analysis of the remaining peptides’ physicochemical properties revealed multiple features that contribute either positively or negatively to back exchange discrepancies. Employing our workflow for comparable measurements on synthetic peptide mixtures containing post-translational modifications, and their unmodified counterparts, we show that lysine acetylation has a strong effect on the observed exchange rate, whereas serine/threonine phosphorylation does not. Our automated workflow enables high-throughput determination of exchange rates in complex biological peptide mixtures with diverse properties. • Novel high-throughput HX-MS workflow for calculating k obs across hundreds of peptides. • Majority of k obs falls within a 2-fold range of the k calc predicted values. • Peptide physicochemical traits strongly influence back exchange rates. • Acetylation significantly affects k obs while phosphorylation has minimal impact. • FD controls are essential for comparing modified and unmodified peptides.

  PublisherDOI

• Comprehensive Internal Standards for Hydrogen–Deuterium Exchange Mass Spectrometry

  Analytical Chemistry · 2025-10-27 · 1 citations

  articleSenior authorCorresponding

  Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a widespread tool for studying protein structure and dynamics, but data comparability across different experimental conditions, instrument platforms, and laboratories remains challenging. Here, we design, synthesize, and validate a novel set of imidazolium-based internal exchange reporters (IERs) that readily integrate with HDX-MS to provide a direct measure of exchange conditions. The set of eight IERs are designed to have a single discrete exchangeable site to sample conditions over exceptionally broad time scales, from milliseconds to days, while also exhibiting desirable chromatographic behavior. We demonstrated the utility of these IERs and the associated computational tools through a series of comparative HDX-MS studies involving well-studied proteins where reaction conditions like pH and deuterium content were offset. Through unambiguous referencing of exchange reaction conditions, this novel set of internal standards enables rigorous cross-experiment comparisons and will improve HDX-MS-derived models of protein dynamics.

  PublisherDOI

• Mapping Hydrogen Migration Thresholds for Site-Specific HDX-MS

  Molecular & Cellular Proteomics · 2025-10-10 · 2 citations

  articleOpen accessSenior author

  A long-standing limitation of Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) has been the difficulty in accurately measuring amide exchange with single amide resolution. Excitation of peptides or proteins during ionization, ion transmission, or collisional activation rapidly induces intermolecular hydrogen migration, leading to a loss of the deuterium-labeled state; a term commonly known as "scrambling." Electron-based fragmentation methods in conjunction with gentle ion transmission settings can minimize scrambling but often not completely. Levels of scrambling have been shown to vary with ion transmission settings, peptide charge, and size, but the general properties that govern the susceptibility of peptides to scrambling are not well understood. Furthermore, it remains unclear whether scrambling is generally a global process or if local scrambling networks commonly exist within peptides. Here, we examine a panel of peptides using gentle electron transfer dissociation and map the activation thresholds of scrambling to define a relationship between peptide charge density and scrambling propensity. This study suggests that by and large, the scrambling process has a single activation threshold and involves all exchangeable sites within a peptide. For some peptides, the activation energy required for scrambling is surprisingly close to that of amide bond dissociation.

  PublisherDOI

• A type-specific B-cell epitope at the apex of outer surface protein C (OspC) of the Lyme disease spirochete, <i>Borreliella burgdorferi</i>

  Microbiology Spectrum · 2025-02-14 · 1 citations

  articleOpen access

  ABSTRACT Broadly protective immunity to the Lyme disease spirochete, Borreliella burgdorferi , is constrained by antibodies against type-specific epitopes on outer surface protein C (OspC), a homodimeric helix-rich lipoprotein essential for early stages of spirochete dissemination in vertebrate hosts. However, the molecular basis for type-specific immunity has not been fully elucidated. In this report, we produced and characterized an OspC mouse monoclonal antibody, 8C1, that recognizes native and recombinant OspC type A (OspC A ) but not OspC type B or K. Epitope mapping by hydrogen–deuterium exchange mass spectrometry (HDX-MS) localized 8C1’s epitope to a protruding ridge on the apex of OspC A α-helix 3 (residues 130–150) previously known to be an immunodominant region of the molecule. Alanine scanning pinpointed 8C1’s core binding motif to a solvent exposed patch consisting of residues K 141 , H 142 , T 143 , and D 144 . Analysis of 26 Lyme disease-positive serum samples confirmed human antibody reactivity with this region of OspC A , with residues E 140 and D 144 as being the most consequential. Our results underscore the importance of α-helix 3 as a target of type-specific epitopes on OspC A that should be taken into consideration in Lyme disease vaccine design. IMPORTANCE A central challenge in the development of vaccines against Lyme disease, the most common vector-borne infection in the United States, is the antigenically variable nature of the lipoproteins displayed on the surface of the disease-causing spirochete, Borreliella burgdorferi . For example, antibodies against one type of outer surface protein C (OspC), a lipoprotein involved in B. burgdorferi transmission and early stages of infection, may have little or no cross reactivity with another seemingly closely related variant of OspC, thereby hampering the use of a single OspC type as a vaccine antigen. For the sake of vaccine design, it is critical to identify the specific epitopes on OspC that both restrict and enable cross-reactivity.

  PublisherDOI

Recent grants

• Investigating the Architecture of HIV Envelope Glycoprotein

  NIH · $101k · 2012–2014

• Mechanisms of IgM mediated activation of the complement system

  NIH · $2.4M · 2021–2026

• Hybrid structural mass spectrometry for rapid site-specific glycan structural elucidation

  NIH · $1.7M · 2018–2023

Frequent coauthors

• Ellie I. James

  University of Washington

  41 shared

• Abhinav Nath

  University of Washington

  40 shared

• Kelly K. Lee

  University of Washington

  39 shared

• David W. Baggett

  University of Washington

  26 shared

• Joel B. Schachter

  Takeda (United States)

  25 shared

• Edcon Chang

  Takeda (United States)

  25 shared

• Thomas Nixey

  Takeda (United States)

  25 shared

• Karoline Choi

  St. Jude Children's Research Hospital

  25 shared