Seattle, WA
17 - 18 July, 2025 (Thu-Fri)
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About

The annual Cascadia Proteomics Symposium brings together proteomics researchers from the Pacific Northwest region, Washington, Oregon, and British Columbia, to discuss our great science, get to know each other better, share ideas, and foster collaboration within the region. The program includes oral sessions, vendor booths, and poster presentations with appetizers, Northwest brews and wines, and other refreshments to make this a convivial event.

The 2024 symposium was was the best one yet, so we're doing it again in 2025 at the Institute for Systems Biology on July 17-18 (Thu-Fri).


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2025 Program

Thursday, July 17
Friday, July 18
Posters
Sponsor Posters
Gold Sponsors
800
Continental Breakfast
 
830
Session 4: Metaproteomics and Metabolomics
Chair: Brook Nunn (UW)
835
Brook Nunn (UW)
Peptides that can predict Harmful Algal Blooms
Authors
Brook L. Nunn 1, Miranda C. Mudge 1, Michael Riffle 1, Gabriella Chebli 2, Deanna L. Plubell 1, Tatiana A. Rynearson 3, Julia Kubanek 2,4, William S. Noble 1, Emma Timmins-Schiffman 1

Institutions
1 Department of Genome Sciences, University of Washington, Seattle, WA, USA. 2 School of Biological Sciences, Georgia Institute of Technology, Parker H. Petit Institute for Bioengineering and Bioscience, Atlanta, GA, USA. 3 Graduate School of Oceanography, University of Rhode Island, Kingston, RI, USA. 4 School of Chemistry & Biochemistry, Georgia Institute of Technology, Parker H. Petit Institute for Bioengineering and Bioscience, Atlanta, GA, USA.

Abstract
Harmful algal blooms (HABs) pose a growing threat to ecosystems and public health worldwide, yet remain difficult to forecast. In the San Juan Islands of Washington State, USA, coastal waters experience biannual HABs, offering a rare opportunity to study bloom initiation dynamics. Dynamic interactions between algae, including harmful algae, and bacteria play a large role in regulating water chemistry. Free-living bacteria quickly respond to small physical and/or chemical environmental changes by adjusting their proteome. To investigate whether bacterial proteomic shifts precede algal bloom events, we conducted a high-resolution temporal field study, sampling the free-living microbiome in Eastsound, WA every 4 hours for 22 days in June 2021. To characterize the microbiome’s physiological changes preceding bloom onset, we collected and analyzed a high-resolution metaproteomic time series of a free-living microbiome in a coastal ecosystem. We hypothesized that this response is detectable at the peptide level and occurs before the rapid phytoplankton growth characteristic of harmful bloom events. We developed an agnostic, comprehensive, and statistically robust framework to query the microbial metaproteome for bacterial peptides that undergo a non-reverting state shift in abundance approximately 24 hours prior to HAB onset. The microbiome’s metaproteome was analyzed on a Lumos mass spectrometer using Data Independent Acquisition (DIA) and processed with Prosit, EncyclopeDIA, and Skyline. Resulting spectra were searched against a time- and location-specific metagenome to identify and quantify peptides from the DIA data. A multistep framework was developed to select and statistically validate candidate biomarker peptides using a hidden Markov model (HMM), a time series approach capable of detecting non-reverting state shifts in quantified features. To our knowledge, this represents the first application of HMM-based time series modeling to metaproteomic data. Our investigation revealed 314 potential biomarkers for predicting bloom onset. A subset was then selected to determine limits of quantification (LOQ) and detection (LOD) using Matrix-Matched Calibration Curves (MMCC). Targeted validation of detectable and quantifiable peptides was then tested on a second pre-bloom period with a different chemical profile. Twelve peptides were confirmed to be quantifiable, non-reverting biomarkers that can predict the onset of blooms at this site. These findings support the use of the bacterial microbiome to generate biomarkers for predicting future HABs and highlight the broader potential of microbiome analysis for anticipating other host-related outcomes.
900
Shabnam Salimi (UW)
Decoding Aging and Kidney Function: Bayesian Metabolomic Aging and Kidney Aging Clocks
Authors
Shabnam Salimi, Danijel Djukovic, Hayley Purcell, Daniel Raftery

Institutions
Department of Anesthesiology and Pain Medicine, North West Metabolomic Center, University of Washington

Abstract
Background: Aging occurs heterogeneously across organs and individuals. The kidney, a central organ in metabolic homeostasis, exhibits variable decline with age. However, early biomarkers of kidney aging and decline remain poorly defined. Objective: We aimed to develop set of metabolomic clocks—namely a Metabolomic Aging Clock, a Metabolomic Kidney Function Clock, and a Metabolomic Kidney Aging Clock—using Bayesian variable selection and plasma metabolomics in a relatively healthy population. Methods: We analyzed plasma samples from 750 individuals (mean age 48.5 ± 12.2 years) free of overt kidney disease, recruited from three clinical sites. Metabolomic profiling was conducted using high-resolution mass spectrometry. Kidney function was quantified via eGFR using the CKD-EPI creatinine equation. We applied a Bayesian framework with projection predictive variable selection and LASSO to identify metabolite features predictive of chronological age, kidney function (eGFR), and Aging Kidney. Horseshoe priors were used for sparsity, and model performance was evaluated via Pareto-smoothed importance sampling Leave-One-Out Cross-Validation (PSIS-LOO-CV). Results: We identified distinct metabolite panels for each clock. For the Metabolomic Aging Clock, key features included phenylacetylglutamine, pantothenate, acetyl carnitine, Hydroxy isovaleric Acid, N-acetyl-aspartyl-glutamate, and cystine. The Metabolomic Kidney Function Clock was characterized by pseudouridine, pyridoxic acid, deoxycarnitine, and aminoadipate, among others. The Metabolomic Kidney Aging Clock integrated eGFR-age residuals and revealed predictive metabolites such as Aminoadipate, Hydroxybenzoic Acid, histidine, methylhistidine, dimethylglycine, and Kynurenic Acid. ROC-AUC analyses confirmed superior performance of the kidney-specific clocks in identifying individuals with CKD (eGFR <60 ml/min/1.73m²).

Conclusion: Using machine learning and Bayesian inference, we developed robust and interpretable metabolomic clocks that quantify systemic and kidney-specific aging. These tools offer novel biomarkers for early detection of kidney decline and support the concept that early, subclinical organ dysfunction contributes to whole-body aging entropy. Our findings provide a foundation for precision aging assessments.
920
Jinyu Liu (UW)
Exploring the Feasibility of MS³ for Accurate Quantitation of Endogenous Retinoids in Complex Biological Matrices
Authors
Jinyu Liu, Nina Isoherranen

Institutions
Department of pharmaceutics, University of Washington

Abstract
Retinoic acids (RAs), particularly all-trans retinoic acid (atRA), are essential endogenous signaling molecules that regulate development, immune function, and lipid metabolism. Their quantitation in biological matrices is key to understanding retinoid homeostasis in both health and disease. However, their low abundance, chemical lability, and endogenous nature complicate bioanalysis. We previously developed a robust liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to characterize the human hepatic vitamin A metabolome, using liquid–liquid extraction (LLE) or protein precipitation for sample preparation and deuterated isotopes (RA-d₅) as the internal standard for normalization. This method has been applied to quantify retinoids in various complex biological samples. Analyses were conducted on a Sciex 5500/6500 QTRAP mass spectrometer with atmospheric pressure chemical ionization, with multiple reaction monitoring (MRM) transitions (e.g., 301→205 for retinoic acids and 306→208 for isotopes) for analyte quantitation. As matrix-derived interferences often persist even after extraction, in parallel to the MRM, MS³ (MS/MS/MS, second-generation product ion monitoring) data with 301→205→159 transitions were collected to confirm peak identity of RA isomers.

Retinoid quantitation is complicated by their endogenous nature, which precludes the use of true blank matrices. This makes it difficult to distinguish matrix-specific artifacts and interferences observed occasionally as shoulders and double peaks in chromatograms from true analyte signals. MS³ provides a higher degree of filtering by adding a third stage of fragmentation and isolating a secondary product ion, thereby improving selectivity and reducing background interference.

The goal of this project was to assess the feasibility of MS³-based quantitation of atRA and 13cisRA. To test for linearity of the MS3 response, standard curves of atRA and 13cisRA (1–200 nM), spiked into charcoal-treated blank human serum and extracted using LLE, were analyzed by both MRM and MS³ modes. The peak area ratio to the internal standard (180 nM) was used to normalize signal.

There were no statistically significant differences in measured concentrations between the two modes across most of the calibration range. At commonly observed tissue concentrations (above 5 nM), the deviations between modes were generally <10%, suggesting good agreement. At lower concentrations, particularly near the lower limit of quantitation (<1 nM), the differences in some cases exceeded 30%. Nevertheless, even at the low QC level (<5 nM), the difference remained within 10–15%, supporting the practical utility of MS³ for typical biological applications.

Importantly, MS³ chromatograms consistently showed improved signal clarity compared to MRM, especially in matrices prone to co-eluting interferences. Our preliminary results demonstrated both sufficient sensitivity and quantitative accuracy of MS³, supporting the feasibility of using MS³ not only as a confirmatory tool but also as a reliable quantitation mode under matrix-compromised conditions.
940
Lightning Talk: Wentao Zhu (UW)
Low Glucose and SCD1 Inhibition Impairs Cancer Metabolic Plasticity and Growth in Cancer Cells: A Comprehensive Metabolomic and Lipidomic Analysis
Authors
Wentao Zhu1,2, John A. Lusk1,2, Vadim Pascua1,2, Danijel Djukovic1,2, Daniel Raftery1,2,3*

Institutions
1 Northwest Metabolomics Research Center, and 2 Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, 3 Fred Hutchinson Cancer Center, Seattle, WA 98109

Abstract
Background: Cancer cells exhibit remarkable metabolic plasticity, enabling them to adapt to fluctuating nutrient conditions. This study investigates the impact of a combination of low glucose levels and inhibition of stearoyl-CoA desaturase 1 (SCD1) using A939572 on cancer metabolic plasticity and growth. Methods: A comprehensive metabolomic and lipidomic analysis was conducted to unravel the intricate changes in cellular metabolites and lipids. MCF-7 cells were subjected to low glucose conditions, and SCD1 was inhibited using A939572. The resulting alterations in metabolic pathways and lipid profiles were explored to elucidate the synergistic effects on cancer cell physiology. Results: The combination of low glucose and A939572-induced SCD1 inhibition significantly impaired cancer cell metabolic plasticity. Metabolomic analysis highlighted shifts in key glycolytic and amino acid pathways, indicating the cells’ struggle to adapt to restricted glucose availability. Lipidomic profiling revealed alterations in lipid composition, implying disruptions in membrane integrity and signaling cascades. Conclusion: Our findings underscore the critical roles of glucose availability and SCD1 activity in sustaining cancer metabolic plasticity and growth. Simultaneously targeting these pathways emerges as a promising strategy to impede cancer progression. The comprehensive metabolomic and lipidomic analysis provides a detailed roadmap of molecular alterations induced by this combination treatment, that may help identify potential therapeutic targets.
945
Lightning Talk: Keiann Simon (UW)
Oxidation of Δ9-THC and Δ8-THC by CYP2C9, CYP2C19, and CYP3A4 Reveals Regiospecific Metabolite Formation and CYP Structure-Function Relationships
Authors
Keiann T. Simon, Gayatri Kundassery, Michal Maes, Nina Isoherranen

Institutions
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, USA

Abstract
The plant cannabis sativa, recognized for its diverse therapeutic and pharmacological effects, contains over 100 cannabinoids, of which delta-9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the most extensively studied.1 These cannabinoids interact with the endocannabinoid system, influencing physiological processes such as pain, mood, appetite, and memory.2,3 Among the lesser-studied cannabinoids, delta-8-tetrahydrocannabinol (Δ8-THC) has recently gained interest following the legal changes brought about by the 2018 Farm Bill, which recognized hemp-derived cannabinoids as legal. This study aimed to define the regiospecific metabolism of Δ9-THC and Δ8-THC by CYP enzymes, and to assess whether Δ9-THC metabolic pathways can predict Δ8-THC metabolism. Using LC-MS/MS (AB SCIEX 5500 QTRAP) based metabolite identification and in vitro incubations with human liver microsomes (HLMs) and recombinant CYP enzymes, we identified key sites of oxidation and enzyme-specific metabolites for both cannabinoids. CYP2C9 selectively oxidized Δ9-THC at carbon 11 (C-11) to form 11-hydroxy-Δ9-THC (11-OH), a pharmacologically active metabolite, and further oxidized it to 11-carboxy-Δ9-THC (11-COOH). CYP2C19 formed both 11-OH and 8α-hydroxy-Δ9-THC (8α-OH), while CYP3A4 predominantly produced 8α-OH and 8β-hydroxy-Δ9-THC (8β-OH) without generating 11-OH. Sequential metabolism of the primary metabolites of Δ9-THC also revealed conserved regiospecificity where the oxidation of 8α-OH and 8β-OH at C-11 by CYP2C9 and CYP2C19, yielded di-hydroxylated products tentatively identified as 8α,11-OH-Δ9-THC and 8β,11-OH-Δ9-THC, while CYP3A4 oxidized 11-OH only at C-8 to form both diols. No diols were observed in CYP2C9 incubations of 11-OH. Comparative LC-MS/MS based metabolic profiling of Δ8-THC showed overlapping CYP specificity and similar regiospecific metabolites. Molecular modeling and ligand-CYP binding studies were done to support these findings, which elucidated a structure-function relationship that governs regiospecific hydroxylation of CYPs. Altogether, these results demonstrate that both Δ9-THC and Δ8-THC exhibit CYP enzyme-specific regiospecificity in oxidative metabolism. This study also shows LC-MS/MS based analysis can be used to assess human cannabinoid metabolome.

References: 1. Pacher, P., Bátkai, S., & Kunos, G. (2006). The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacological Reviews, 58(3), 389-462. 2. 2. Alger, B. E. and Kim, J. (2011). Supply and demand for endocannabinoids. Trends in Neurosciences, 34(6), 304-315. 3. Watt, G. and Karl, T. (2017). In vivo evidence for therapeutic properties of cannabidiol (CBD) for alzheimer's disease. Frontiers in Pharmacology, 8.
950
Lightning Talk: Michal Maes (UW)
Novel adductomics methods allow identification of Dicloxacillin-protein adduct in human plasma and liver tissue
Authors
Michal Maes1, Jiayao Chen1, Alex Zelter2, Michael Hoopmann1, Daniel Jaschob2, Michael Riffle2, Michael J. MacCoss2, Nina Isoherranen1

Institutions
1 Department of Pharmaceutics and 2 Department of Genome Sciences, University of Washington, Seattle, WA

Abstract
Dicloxacillin is a β-lactam antibiotic associated with a high incidence of drug-induced hypersensitivity and cholestatic liver injury. Although the mechanisms remain unclear, both non-immune- and immune-mediated mechanisms may be involved. One proposed mechanism involves the formation of covalent adducts between dicloxacillin and lysine residues of proteins, where the resulting adducts act as haptens, potentially triggering an immune response. Thus, we hypothesized that dicloxacillin can covalently bind to proteins in plasma and in liver, therefore leading to toxicity. The aim of this study was to identify dicloxacillin-modified peptides and amino acid residues in complex matrices. Specifically, we used our adductomics workflow to detect dicloxacillin-modified peptides in a variety of samples, to try to elucidate the selective toxicity observed in the clinic. We first incubated the dicloxacillin with purified albumin to determine which residues become adducted in the simplest systems. Human albumin has 59 lysines and we detected dicloxacillin adducts on almost all of them. Next, we tested whether these same lysine residues were adducted after in vitro dicloxacillin-treatment of plasma from healthy volunteers (n=6). Several more adducted proteins were found with a high variability between the different donors in these samples. This data suggests that in vitro incubations can be used to predict adduct formation in vivo after exposure to adduct forming xenobiotics. As dicloxacillin results in liver toxicity, we further tested whether adducts in human liver proteins could be identified using our adductomics workflow. HLMs (human liver microsomes) from three donors were incubated with dicloxacillin and DDA and DIA data were collected for adduct identification. This was further investigated by in vitro activity assays for the two main drug-metabolizing enzymes in the liver, CYP2C9 and CYP3A4. Our results show the strength of combining a range of simple protein-only samples to more biologically complex and donor-specific samples when looking for adducted peptides and proteins. For the dicloxacillin, this might shed light on the degrees of toxicity observed in individual patients.
955
Break
 
1025
Session 5: Technologies and Applications
Chair: Dan Raftery (UW)
1030
Tai-Tu Lin (PNNL)
Antibody Cocktail-Based Immunoaffinity Enrichment and LC-SRM for Multiplexed Quantification of Circulating Proinsulin Proteoforms and C-Peptide
Authors
Wei-Jun Qian, Qingqing Shen, Wang Cao, Jun Qu, Carmella Evans-Molina, Emily K. Sims

Institutions
Biological Sciences Division, Pacific Northwest National Laboratory; The Department of Pharmaceutical Sciences, University at Buffalo; The Center for Diabetes & Metabolic Diseases, Indiana University School of Medicine

Abstract
Insulin plays a crucial role in regulating blood glucose levels and maintaining metabolic homeostasis. It is initially synthesized as proinsulin, a precursor that undergoes sequential processing to form two intermediate proteoforms: des-31,32 proinsulin and des-64,65 proinsulin. These intermediates are further processed to generate mature insulin and C-peptide.

Persistent proinsulin secretion and elevated proinsulin-to-C-peptide ratios have been observed in individuals with type 1 diabetes (T1D). However, accurately quantifying proinsulin proteoforms in circulation remains challenging due to their high sequence and structural similarities, which make differentiation difficult using conventional immunoassays. Additionally, antibodies used in commercial ELISA kits for proinsulin often exhibit significant cross-reactivity with des-forms of proinsulin, leading to substantial overestimation of proinsulin levels in clinical samples. In this study, we developed a highly sensitive LC-MS-based strategy for the quantification of intact proinsulin, des-31,32 proinsulin, des-64,65 proinsulin, and C-peptide in circulation. Our approach integrates: i) Quantitative and robust affinity capture using an optimized antibody cocktail, which eliminates the quantitative bias across proteoforms observed with single-antibody strategies, ii) ii) Lys-C digestion to generate unique surrogate peptides for each proteoform, and iii) Trapping-nano-LC coupled with FAIMS/dCV-MS for ultra-sensitive analysis. The assay achieved exceptional sensitivity, with serum limits of quantification (LOQs) of 1.7, 2.3, and 3.6 pg/mL for intact proinsulin, des-31,32 proinsulin, and des-64,65 proinsulin, respectively.

To demonstrate the clinical application of this assay, serum samples from at-risk individuals, new-onset T1D subjects, and age- and sex-matched controls (n=20 per group) were analyzed. Across the samples, median levels were 915 pg/mL for C-peptide, 23.1 pg/mL for intact proinsulin, 46.2 pg/mL for des-31,32 proinsulin, and 4.6 pg/mL for des-64,65 proinsulin. The ratios of all three proinsulin proteoforms to C-peptide were significantly higher in T1D subjects compared to controls, although no significant differences were observed between at-risk individuals and controls. Further studies are underway to assess the prognostic value of these markers using larger sample sets. In conclusion, our assay achieved quantification limits in the 1–5 pg/mL range, offering superior sensitivity and specificity compared to clinically approved proinsulin assays. Moreover, all three proinsulin proteoforms provided promising and distinctive features for differentiating T1D subjects from matched controls.
1050
Yue (Winnie) Wen (UW)
Development and Validation of a Targeted LC-MS/MS Method for Liver Fatty Acid Binding Protein (FABP1) Quantification
Authors
Yue Winnie Wen, Nina Isoherranen

Institutions
University of Washington, Seattle

Abstract
Liver fatty acid-binding protein (FABP1) is a 14 kDa soluble protein, playing a key role in the intracellular transport of long-chain fatty acids and other lipophilic molecules. Studies have shown that FABP1 expression level is associated with various disease states. For example, FABP1 is upregulated in the liver of patients with non-alcoholic fatty liver disease or obesity and decreased in rare human genetic lipid malabsorption syndromes. Many drugs, such as diclofenac and flurbiprofen, can also bind to FABP1 and expression level of FABP1 can modulate drug distribution into the liver. Thus, it is important to determine the concentrations of FABP1 in the liver and to understand the inter-individual variability in FABP1 expression. To determine the interindividual variability in FABP1 expression in humans and to establish the organ-specific expression pattern, a novel targeted LC-MS/MS method was developed using selected surrogate tryptic peptide quantification with AB Sciex 5500 QTRAP. The assay was validated, with inter-day, intra-day, and instrument reproducibility all within a 15% coefficient of variation. FABP1 expression in liver tissues of 61 donors with different age, sex, and liver pathology from the UW human liver bank was quantified. In addition, FABP1 expression in limited number of available small intestine (n = 5) and kidney (n=4) tissues were measured. The resulting protein quantification data was used to populate a mathematical model to allow predictions of drug distribution into the tissues in individual patients and on a population level.
1110
Addison Roush (UW)
SLIMPHONY: A New SLIM-Based Instrument That Orchestrates Complex Ion Mobility – Mass Spectrometry Experiments
Authors
AnneClaire Wageman, Addison E. Roush, Yuan (Bruce) Feng, and Matthew F. Bush

Institutions
Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195, United States

Abstract
The inherent heterogeneity of biological macromolecules offers a unique challenge for analysts. The combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) is sensitive to the size, shape, and dynamics of, for example, proteins and their complexes. Combining multiple dimensions of ion mobility and mass spectrometry (IM-IM-MS) while leveraging unique gas-phase ion chemistry between dimensions can increase the information content of protein structural studies. Here, we introduce a new IMS instrument, SLIMPHONY, built using the Structures for Lossless Ion Manipulations (SLIM) architecture. SLIMPHONY is unique in that eight independently controlled traveling-wave regions work in concert to enable complex, multidimensional separations.

Separation of a mixture of proteins and protein complexes is demonstrated through a single-dimension IM-MS experiment, and in accordance with theory, the peak-to-peak resolution of commercial, small-molecule, tuning mix ions is found to increase roughly with the square root of the separation length. When these same commercial tuning mix ions are used as calibrants, SLIMPHONY yields accurate, within 1.16% of literature values, collision cross sections for the peptide bradykinin. Together, these experiments show that SLIMPHONY performs well for a range of analyte classes including native proteins.

In harmony with prior work, ion selection and trapping between dimensions probes the gas-phase unfolding of a subpopulation of native-like ubiquitin ions. By varying the duration of trapping, ion lifetimes ranging from 5 ms to 525 ms have been probed with greater unfolding observed for longer trapping durations. Ions were also collisionally activated between dimensions, which represents an additional pathway to study protein unfolding on SLIMPHONY. Ion structural ensembles generated by collisional activation are preserved as the separation length is extended from 0.87 m up to 4.31 m through a multiple-pass separation demonstrating that there is little evidence for isomerization during separation. With multiple on-board techniques to control the extent of ion activation and variable separation length, SLIMPHONY represents a flexible platform for conducting novel studies of protein structure and unfolding.
1130
Jianji Chen (Just-Evotec)
DOE-Based Comparative Analysis of MAM on Thermo Fisher Q Exactive™ HF and Exploris™ 240 for Therapeutic Antibodies
Authors
Jianji Chen, Erin Weisenhorn, Rosalynn Molden

Institutions
Just-Evotec Biologics

Abstract
Introduction The multi-attribute method (MAM) is increasingly employed in the biopharmaceutical industry for comprehensive characterization of therapeutic antibodies. Despite advancements in mass spectrometry instrumentation, systematic comparisons of performance across platforms remain scarce . This study evaluates two widely utilized instruments, Q Exactive™ and Exploris™ 240, focusing on their capability to identify and quantify post-translational modifications (PTMs) under a design of experiments (DOE) framework. Our findings address a critical gap in MAM workflows, particularly in understanding instrument-specific performance differences in cross-site or cross-instrument data comparisons.

Methods A DOE approach was utilized to assess the performance of Q Exactive™ HF and Exploris™ 240 for MAM workflows. An IgG1-type therapeutic antibody was prepared and analyzed under varying parameter settings, including MS1 AGC, MS1 max injection time, dynamic exclusion, and Top N. Data analysis focused on identification and quantitation of PTMs, such as C-/N-terminal variants, isomerization, deamidation, oxidation, glycation and glycans. Peptide- and precursor-level data were extracted using Protein Metrics’ Byos™ and Skyline (open-source client app) then analyzed with JMP® to evaluate instrument performance across conditions.

Preliminary Data The comparative study revealed that the Q Exactive™ and Exploris™ instruments demonstrate comparable performance in identifying and quantifying common PTMs in therapeutic antibodies. Across varied parameter settings, the instruments achieved consistent results with minimal variance in quantitation accuracy and sensitivity. Key insights include: • Parameter Optimization: Lower MS1 AGC provided more consistent mass accuracy. A dynamic exclusion of 7.5 seconds was found optimal for Q Exactive™. Top 5 settings resulted in higher points per peak (PPP) and more peptide spectrum matches and higher identification scores compared to Top 10. • Instrument Performance: The Exploris™ shorter duty cycles enabled more peptide spectral matches per species and improved identification scores but quantitation remained consistent between the two instruments. • PTM Identification: PTMs above 0.5% could be identified with high confidence in both instruments, supporting their reliability in MAM workflows.

• PTM Quantitation: PTM quantitation was highly comparable between the two instruments (R-square > 0.98) for PTMs greater than 0.5% XIC ratio. Across varied parameter settings, results were consistent with minimal variance in quantitation accuracy and sensitivity. This study underscores the importance of a systematic evaluation to optimize MS data acquisition settings. By leveraging a DOE framework, our findings provide a robust foundation for optimizing MAM methods in therapeutic antibody development. Additionally, the results show these optimized parameters are crucial for consistent characterization of PTMs across sites and instruments.

Novel Aspect A systematic MAM evaluation of Q Exactive™ and Exploris™ using a DOE-based approach to optimize interdependent mass spec parameters efficiently.
1150
Lightning Talk: Chris Weir (UW)
Real-Time Disulfide Bond Reduction Enabled by Programmed-Temperature Electrospray Ionization (ptESI) and the Inconvenience of Electrochemistry during Electrospray
Authors
Christopher J. Weir, Theresa A. Gozzo, Lilly R. Woerner, May A. Constabel, Meagan M. Gadzuk-Shea, and Matthew F. Bush

Institutions
University of Washington

Abstract
Introduction Disulfide bonds affect the structure, stability, and function of proteins. The loss of native disulfide bonds through cleavage and/or scrambling can lead to many negative outcomes, including the loss of native structures, decreased selectivity, decreased activity, and increased aggregation rates. Many protocols for mass spectrometry (MS) include the reduction of disulfide bonds, e.g., samples in bottom-up proteomics are usually reduced and alkylated to prior digestion and samples of antibodies are often reduced to enable mass analysis of the constituent heavy and light chains. The reduction of disulfide bonds is usually performed offline prior to MS analysis and to completion. Here, we control this reaction using temperature and monitor the products in real-time using native MS. Also discussed are several factors contributing to variability in the reduction measurement and ways of improving them.

Methods We have developed a programmed-temperature electrospray ionization (ptESI) source. The ptESI source has a low thermal mass and modulates the temperature of liquid samples as a function of time according to user-defined temperature programs. We characterized this source and found that liquid samples could be heated or cooled with high fidelity at rates exceeding ±30 °C⸱min–1. The source was positioned in front of a Waters Cyclic IMS system to enable the real-time characterization of ions using a variety of MS-based experiments. Samples of ribonuclease A and selected antibodies were prepared in aqueous 200 mM ammonium acetate at pH 7 with 5 mM dithiothreitol (DTT).

Preliminary Data Ribonuclease A was chosen as a test case due to its four native disulfide bonds and strong thermal stability. Samples of ribonuclease A containing DTT were run through a temperature profile that went from 25 to 75 °C at 60 °C⸱min–1, during which time the sample was continuously analyzed by ESI-MS. The initial mass spectrum was consistent with the expected mass of the disulfide-intact protein; that spectrum persisted until 75 °C. The temperature of the sample was then held constant at 75 °C and the mass spectrum showed signs of reduction. The time at which this occurs can be ~± 30 s, and several factors such as pH, temperature, and electrochemistry are discussed as possible reasons for this large variation in reduction times.

NISTmAb was chosen as a starting antibody due to its position as a well characterized standard for antibody testing. Samples of the monoclonal antibodies were prepared with DTT and placed in the ptESI source. They were heated from 10 to 70 °C at 30 °C⸱min–1 and then held at 70 °C with continuous analysis throughout. The products of reduction appear as a function of time. For example, signals for intact NISTmAb decreased as temperature increased, whereas signals for half mAbs (consisting of one heavy and one light chain, most predominant), isolated lights chains, and isolated heavy chains appeared. At later times, the signals for isolated light chains continued to increase, whereas those for intact NISTmAb and half mAbs decreased. These results suggest that NISTmAb might follow a pathway of first breaking into half mAbs, followed by further degradation into individual light and heavy chains.
1155
Lightning Talk: Sara Gosline (PNNL)
From spatial proteomics to spatial multiomics: leveraging spammR to integrate, analyze, and interpret mass spectrometry imaging data
Authors
Yannick Mahlich, Marija Veličković, Jason McDermott, Kim Huffman, Brian Andonian, Gina Many, Paul Piehowski, Sara Gosline

Institutions
Pacific Northwest National Laboratory

Abstract
Advances in mass spectrometry (MS) instrumentation, ultrasensitive sample handling protocols, and data independent acquisition methods have led to massive increases in the sensitivity of MS-based proteomics measurements, pushing the analysis inputs down to the single-cell level(Ctortecka and Mechtler).

These improvements have also fueled the development of spatially-resolved proteomics methods, where biological samples can be sectioned via laser capture microdissection (LCM) into discrete ‘voxels’ that are then analyzed via MS-based proteomics, and the resulting proteomes mapped back to their origin in the tissue (Piehowski et al.). This novel approach has motivated advances in computational methods, specifically the ability to identify proteins that are differentially expressed across a given tissue of interest (Gosline et al.), as well as the ability to overlay measurements of metabolomic and lipidomic measurements from MALDI MS (Veličković et al.).

In this work, we show how spatial integration of metabolomics and proteomics measurements in a spatial context can lead to novel biological hypotheses regarding mechanisms of skeletal muscle atrophy and dysfunction in vastus lateralis muscle biopsies from rheumatoid arthritis patients and matched healthy control subjects(Huffman et al.). Specifically, we measure metabolomics and proteomics in skeletal muscle samples from 11 rheumatoid arthritis patients and compare regions of immune infiltrate between both healthy and diseased myofibers to identify the specific immune signaling pathways active in the disease samples. We describe our new R package, called spammR(Mahlich et al.), and show how it can examine differential expression analysis, rank based enrichment analysis, and correlation based analysis to identify specific metabolite-protein networks that are active across different regions. By comparing pathways active in the disease muscle compared to healthy myofibers we can link these to expression patterns observed in the immune infiltrate.

In summary, we show the power of spatial multiomic integration through our spammR analysis tool applied in a novel rheumatoid arthritis dataset.

Ctortecka, Claudia, and Karl Mechtler. Analytical Science Advances, vol. 2, no. 3–4, 2021, pp. 84–94. https://doi.org/10.1002/ansa.202000152. Gosline, Sara JC, et al. Molecular & Cellular Proteomics, vol. 22, no. 8, 2023. Huffman, Kim M., et al. Arthritis Research & Therapy, vol. 19, no. 1, Jan. 2017, p. 12. Springer Link, https://doi.org/10.1186/s13075-016-1215-7. Mahlich, Yannick, et al. PNNL-CompBio/spammR: V0.01. beta, Zenodo, 16 May 2025. Zenodo, https://doi.org/10.5281/zenodo.15442720. Piehowski, Paul D., et al. Nature Communications, vol. 11, no. 1, Jan. 2020, p. 8. https://doi.org/10.1038/s41467-019-13858-z. Veličković, Marija, et al. Nature Communications, vol. 16, no. 1, Feb. 2025, p. 2061. https://doi.org/10.1038/s41467-025-57107-y.
1200
Lightning Talk: Vicky Sun (UW)
Impact of Enteroid Culture Methods on Nutrient and Xenobiotic Transporter Expression
Authors
Vicky L. Sun, Kai Wang, Edward J. Kelly, Samuel L.M. Arnold

Institutions
Department of Pharmaceutics, University of Washington, Seattle, WA 98195; Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98195

Abstract
Enteroids are in vitro models derived from intestinal tissue with promising characteristics such as multiple epithelial cell types and crypt- and villus-like morphology. The relevance of enteroids as models for xenobiotic and nutrient transport studies remains unclear due to a lack of data on transporter protein expression in these models. Enteroid culture format (e.g., 3-dimensional or monolayer) has been reported to influence transporter mRNA abundance. For this project, we investigated the protein expression of nutrient and xenobiotic transporters in enteroids cultured as monolayers on either 96-well plates or Transwell inserts. Transporter protein expression was compared among enteroid monolayers on 96-well plates, enteroids on Transwell inserts, and paired small intestine mucosa. Mucosa and enteroid proteins were digested using a single-pot, solid-phase enhanced sample preparation (SP3) protocol and analyzed by data-independent acquisition mass spectrometry (DIA-MS).

Initial results suggest overall that enteroids express similar nutrient and xenobiotic transporters as small intestine mucosa, regardless of enteroid monolayer culture format. Among four pediatric donors (n = 3 experimental replicates), ~100 nutrient and xenobiotic transporters were identified in mucosa and enteroids cultured on 96-well plates or Transwell inserts. After normalization to total ion chromatogram (TIC) area and villin-1 protein peak area, several nutrient transporters had similar protein peak areas in both mucosa and enteroid monolayers. However, the basal monosaccharide transporter GLUT2 (SLC2A2) and the apical vitamin C transporter SVCT1 (SLC23A1) were only detected in mucosa, and the glucose transporter GLUT1 (SLC2A1) had ≥ 7-fold higher protein peak area in enteroid monolayers on 96-well plates and ≥ 13-fold higher area in enteroid monolayers on Transwell inserts relative to mucosa. The majority of xenobiotic transporters were expressed in both mucosa and enteroid monolayers, but had slightly higher villin-normalized protein abundance in enteroid monolayers than mucosa, and in enteroid monolayers on Transwell inserts than monolayers on 96-well plates. These initial results suggest enteroids are broadly physiologically relevant as models of the human small intestine mucosa, but have differential expression of specific transporters that should be considered for future functional studies.
1205
Lightning Talk: Lucas Narisawa (UW)
Photo-Crosslinking for Identifying Residue-Level Interactions Involving Proteins with Intrinsic Disorder
Authors
Lucas Narisawa1, Lindsey D. Ulmer1, Matthew F. Bush1 Lucas Murray2, Chip L. Asbury2 Mia Cervantes3, Jasleen K. Sidhu3, Maria K. Janowska3, Rachel E. Klevit3

Institutions
1 Department of Chemistry, University of Washington, Seattle, WA 98195, United States 2 Department of Physiology & Biophysics, University of Washington, Seattle, WA 98195, United States 3 Department of Biochemistry, University of Washington, Seattle, WA 98195, United States

Abstract
Intrinsically disordered regions (IDRs) are prevalent throughout the human proteome but are largely intractable to structural biology techniques due to their many transient or plastic interactions. Small heat shock proteins (sHSPs) are holdases for disease-associated, aggregation-prone proteins and contain a disordered N-terminal region (NTR) that is key for their protein-protein interactions. To probe the NTR interactions of sHSPs, we integrate a photoreactive crosslinking amino acid, benzoyl-L-phenylalanine (BPA), into specific NTR sites of sHSPs. Through crosslinking, tandem mass spectrometry, and bioinformatics, we have identified ensembles of interactions between sHSPs and tubulin proteins, which are are aggregation-prone clients. We propose that this strategy will be useful in a wide range of protein systems containing intrinsic disorder.
1210
Lightning Talk: Anna Lin (UW)
Programmed-Temperature Electrospray Ionization (ptESI): A New Strategy for Characterizing Protein Stability.
Authors
Anna B. Lin, Matthew F. Bush

Institutions
University of Washington

Abstract
Introduction: The Bush Lab has developed a programmable-temperature electrospray ionization (ptESI) source capable of modulating the temperatures of liquid samples across varying gradients and enabling real-time mass spectrometry (MS) analysis. This technology has enabled us to probe protein folding, protein unfolding, which are sensitive to protein stability and useful applications in drug development and quality control. Here, we demonstrate the potential of the ptESI source for the characterization of transthyretin (TTR), a homotetrameric protein implicated in familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA). TTR tetramer can destabilize and dissociate into individual monomers, which are prone to misfolding and aggregation into amyloid fibrils that will develop into these amyloid diseases. A promising strategy to combat this process involves the use of pharmacological chaperones, which are small molecules designed to bind to their target protein and stabilize the native tetrameric form of TTR, and in turn, reduce the likelihood of misfolding and aggregation.

Methods: Tetrameric transthyretin (13 uM) solutions were prepared in a blend of 200 mM ammonium acetate and 200 mM acetic acid at pH 7 and pH 4. Samples were further exchanged using Bio-Rad BioSpin columns and loaded into the ptESI source using nano-ESI borosilicate capillaries. Temperature programs spanning 20 °C to 90 °C with various gradients were used to modulate the temperatures of solutions while MS data was continuously acquired using a Waters Cyclic IM-MS system.

Results: For solutions at pH 7, as the solution temperature increased from 20 °C to 90 °C, the charge state distribution shifted toward higher charge states, with the weighted average increasing from 14.8 to 15.8 which is consistent with protein unfolding in solution. After cooling back down to 20 °C, the original charge state distribution appeared on the spectrum once again and the weighted average would return to 14.8, suggesting complete reversibility. This data demonstrates the ability of the ptESI source to track the unfolding and refolding of the TTR tetramer while also showcasing the high thermal stability of wild-type TTR and its ability to withstand a full temperature cycle with no evidence of dissociation or misfolding. However, as wild-type TTR tetramer is not prone to amyloidosis, transthyretin in native conditions is not a good candidate to identify pharmacological chaperones. To observe transthyretin monomer, the component that aggregates and develops into amyloid diseases, the solution was adjusted to pH 4 to destabilize the tetramer and encourage dissociation.

For solutions under acidic conditions (pH 4) with thermal cycling, we begin to see partial dissociation of the tetramer into its monomer subunits at ~75 °C. After a dwell at 75 °C for 10 minutes, the solution was cooled and reassociation of the tetramer was observed ~45 °C with almost full recovery of the tetrameric state. This reversible behavior was reproduced for several consecutive temperature cycles, which suggests a temperature-sensitive equilibrium between the temperature range of 45 °C and 80 °C. Ongoing work aims to continue characterizing this temperature range as well as explore the effects of ligand binding on TTR stability.
1215
Lightning Talk: Rachel Hu (UW)
Defining a minimally representative signaling state through surface-localized phosphosites
Authors
Rachael Hu, Alexis Chang, Ricard Rodriguez-Mias, Matthew Berg, Judit Villén

Institutions
University of Washington

Abstract
Cell surface proteins allow a cell to sense and respond to its environment and interact with neighboring cells. Key classes of surface proteins, such as receptor tyrosine kinases, detect external cues and initiate signaling cascades through reversible phosphorylation . Detecting and quantifying phosphorylation events at the cell surface uncovers the earliest stages of signal transduction and allows us to trace how cells encode and respond to specific stimuli. Despite advances in phosphoproteomics it is still difficult to capture phosphosites at the cell surface. Technical hurdles such as hydrophobicity, glycosylation, and low abundance make membrane-embedded proteins difficult to detect and quantify by standard mass spectrometry approaches. To address these challenges, we devised two enrichment strategies for cell surface proteins. In the first approach, we targeted glycan groups using biotin hydrazide. In the second approach, we used a membrane impermeable NHS-biotin to label the primary amine of membrane proteins. After labeling, membrane proteins were enriched using streptavidin, digested into peptides and phosphopeptides were enriched. Labeling of primary amines displayed the highest efficiency and we observed over 1,300 unique surface proteins, including integrins and low-abundance HLA markers. Subsequent phosphopeptide enrichment from these surface-enriched samples enabled detection of approximately 400 unique phosphosites mapped to surface proteins. Capturing surface proteins offers insight into cellular spatial organization, while identifying surface-localized phosphosites enables the discovery of minimal molecular signatures that define specific signaling states. This framework supports systematic discovery of surface protein phosphorylation events to define a minimally representative signaling state.
1220
Full Catered Lunch
 
1335
Session 6: Post-translational modifications and variants
Chair: Nina Isoherranen (UW)
1340
Kristian Swearingen (ISB)
Elucidating the role of protein glycosylation in the malaria parasite Plasmodium.
Authors
Kristian E. Swearingen1, Priya Gupta2, Vladimir Vigdorovich2, D. Noah Sather2, Ashley M. Vaughan2

Institutions
1) Institute for Systems Biology; 2) Seattle Children's Research Institute

Abstract
Glycosylation of proteins with covalently-linked sugar moieties is a ubiquitous and essential co-translational modification. Until recently, however, it was debated whether protein glycosylation occurred in Plasmodium, the single-celled eukaryotic pathogen responsible for the disease malaria. Using mass spectrometry-based proteomics, we discovered that conserved thrombospondin type-1 repeat (TSR) domains in the parasite can be modified with two rare glycosylations: O-linked fucose at threonine and C-linked mannose at tryptophan. In order to investigate the role of these protein modifications in parasite virulence, we generated transgenic lines of the human malaria parasite P. falciparum wherein O-fucosylation or C-mannosylation is prevented by single-residue substitutions at the glycan attachment sites. We have found that preventing glycosylation of key invasins produces severe defects in parasites’ ability to transmit from the mosquito vector to the vertebrate host. In order to elucidate the mechanisms underlying these phenotypes, we have developed a suite of recombinant protein expression systems and biophysical assays that we are using to determine why certain proteins are selected for glycosylation and how these modifications modulate protein folding.
1400
Haley Schramm (UW)
New approaches in electron transfer dissociation for characterizing the glycocalyx
Authors
Haley M. Schramm1, Tim S. Veth1, Joshua D. Hinkle2, John E. P. Syka2, Graeme C. McAlister2, Christopher Mullen2, and Nicholas M. Riley1

Institutions
1: University of Washington, 2: Thermo Fisher Scientific

Abstract
The glycocalyx is a sugar coat that surrounds all cells and is integral to our understanding of human health and disease. These species, particularly glycoproteins and glycosaminoglycans (GAGs), regulate numerous cell functions such as cellular signaling. Electron transfer dissociation (ETD) is an indispensable tool for sequencing such complex biopolymers. Despite this, lower quality HCD spectra are often favored in high-throughput experiments because the near instantaneous fragmentation of HCD results in faster acquisition relative to ETD. In short, more HCD MS/MS spectra translate to more identifications despite lower quality.

One important parameter for reaction kinetics is ion cloud overlap during ETD reactions, which is governed by the reaction q-values that are set proportional to the reagent ion m/z. The vast majority of ion-ion reactions are conducted with a q-value at 0.4 relative to the fluoranthene reagent anion’s 202 m/z value, generating a low m/z cutoff of ~80 m/z that is suitable for most proteomic applications. Here, we evaluate ion-ion reactions conducted at higher Mathieu q-values relative to 202 m/z, which increases reaction rates without drastic loss in low m/z ions necessary for high-quality MS/MS spectra. In the context of glycoproteomics, we show how shortening the reaction times based on kinetic considerations can generate sufficient product ion signal-to-noise with faster spectral acquisition rates. Ultimately, we demonstrate how these gains translate to improved glycopeptide characterization with ETD-based fragmentation.

Separately, it has been shown that the traditional approach to spraying tryptic peptides in positive electrospray mode may not be optimal for acidic glycopeptides or GAGs. We also describe our recent efforts to utilize negative ETD (NETD) to sequence such analytes.

We first characterized NETD reaction kinetics on the Orbitrap Ascend using glycopeptides and chondroitin sulfate standards. Our preliminary work has demonstrated successful NETD fragment generation using z = -8 to z= -5 precursor charge states from a chondroitin sulfate (CS) 10-mer comprised of 5 disaccharide repeating units. In all, this study will provide fundamental information for optimizing NETD reaction parameters using several classes of biomolecular anions.
1420
Alexis Chang (UW)
R2HaPpY: Rapid-robotic phosphotyrosine peptide enrichment using HaloTag-Src SH2 pY superbinder
Authors
Alexis Chang, Ricard A. Rodriguez-Mias, Matthew D. Berg, Sophie Moggridge, Judit Villén

Institutions
Department of Genome Sciences, University of Washington, Seattle, USA, 98105

Abstract
Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, proteomic studies of tyrosine phosphorylation have been limited in scale and throughput due to the need for specialized enrichment with costly reagents and labor-intensive protocols. To address these challenges, we developed R2HaPpY, a phosphotyrosine enrichment method that combines highly simplified phosphotyrosine superbinder reagent preparation and automated high-throughput enrichment. Our new reagent binds phosphotyrosine peptides at higher efficiency than other enrichment reagents and reduces both cost and preparation time by 20-fold. To showcase the R2HaPpY method, we apply it to study EGF signaling dynamics in HeLa cells. Using only ~1mg of input peptides, we detect and quantify 1,651 unique phosphotyrosine sites. These include 878 regulated pY sites, many of which are low abundance and not previously detected or annotated as EGF-responsive. Our results reveal differential temporal regulation, allow us to investigate signaling pathway organization, and represent the largest phosphotyrosine dataset of cellular response to EGF stimulation to date. This streamlined and sensitive method facilitates comprehensive, quantitative mapping of tyrosine phosphorylation dynamics, enabling broader integration of phosphotyrosine signaling into multiomic and network-level models across diverse biological systems and disease states.
1440
Catherine Sniezek (UW)
Characterization of computationally-designed protein agonists with phosphoproteomics
Authors
Catherine Sniezek, Riya Keshri, Thomas Schlichthaerle, Chris McGann, Chelsea Lin, David Baker, Hannele Ruohola-Baker, Devin Schweppe

Institutions
Catherine Sniezek (Presenter) -- Genome Sciences, University of Washington Riya Keshri -- Department of Biochemistry, University of Washington; Institute for Stem Cell and Regenerative Medicine, University of Washington Thomas Schlichthaerle -- Department of Biochemistry, University of Washington Chris McGann -- Genome Sciences, University of Washington Chelsea Lin -- Genome Sciences, University of Washington David Baker -- Department of Biochemistry, University of Washington; Institute for Protein Design, University of Washington; Howard Hughes Medical Institute, University of Washington Hannele Ruohola-Baker -- Department of Biochemistry, University of Washington; Institute for Stem Cell and Regenerative Medicine, University of Washington Devin Schweppe -- Genome Sciences, University of Washington

Abstract
Introduction Owing to their high specificity and affinity for their targets, protein-based biologic therapies have revolutionized the treatment of previously incurable autoimmune disorders and cancers. Computational protein design has emerged as an approach to biologics development which has resulted in thermally-stable, high-affinity, protein based binders (“minibinders”) which can be designed to either agonize or inhibit a specific biological pathway. Yet, scalable assays which holistically measure the effects that computationally-designed biologics have on the cell are lacking. This work seeks to probe the biologic efficacy of novel, designed agonists with mass-spectrometry based phosphoproteomics, which will positively impact our ability to design proteins with specific biological impacts. Further, because the novel agonists studied here target membrane proteins such as receptor tyrosine kinases, this work will improve current proteomic workflows for the study of membrane proteins.

Methods Various computationally-designed agonists were studied with phosphoproteomics. Cells (CHO, SH-SY5Y, and TF-1) were treated with 100 nM of native agonist or novel designed agonist for 15 minutes. Cells were washed with PBS and lysed, peptides were isolated by SP3 and labeled with TMT, and pooled sample was enriched for phosphopeptides with Fe-NTA beads. LC/MS analysis was performed over several injections, including MSA-CID untargeted approaches, as well as novel PTM-focused real-time search and targeted MS2 methods.

Results Phosphoproteomic analyses effectively characterized the efficacy of four novel agonists: two targeting NTRK1, one bi-specific agonist targeting NTRK1 and BMPR2, and one bi-specific agonist targeting HER2/FGFR. Phosphopeptides were enriched to 91-99% specificity, with final dataset sizes up to 8500 phosphosites. Kinase-set and gene-set enrichments showed the NTRK1 agonists acting through pathways such as AKT and PKC-a. The HER2/FGFR agonist was found to act through AKT and MAPK, and notably induced other surprising phosphorylation events, such as in pyruvate dehydrogenase, indicating that this agonist acts through novel signaling mechanisms. Additionally, targeted mass spectrometry methods were able to capture phosphorylation changes to target receptor phosphosites which the untargeted methods were unable. Taken together, these data demonstrate the unique and effective ways in which novel agonists act on the cell and provide groundwork for future studies of novel proteins.
1500
Matthew Berg (UW)
Uncovering the functional impact of missense mutations proteome-wide using mistranslation and mass spectrometry
Authors
Matthew D. Berg, Alexis Chang, Kyle Hess, Ricard A. Rodriguez-Mias, Judit Villén

Institutions
Department of Genome Sciences, University of Washington, Seattle, WA, USA

Abstract
DNA sequencing has identified millions of natural genetic variants that alter protein sequence. However, determining the functional impact of these variants remains challenging. Traditional mutagenesis approaches are not scalable for millions of variants and high-throughput approaches such as deep mutational scanning are limited to investigating one protein per experiment. Recently, our lab established Miro – a high-throughput proteomic approach to functionally annotate the impact of missense mutations across entire proteomes. In this approach, stochastic errors in protein synthesis are induced to create amino acid substitutions throughout all expressed proteins within a cell. Biochemical selections that probe general protein properties like solubility, thermal stability, ligand binding, protein-protein interactions and post-translational modifications are then applied to the collection of protein variants. After selection, variants are quantified by mass spectrometry to determine the functional impact of each mutation on the measured property. Here, we harness a collection of tRNAs that we engineered to mis-incorporate alanine at non-alanine codons and determine the impact of over 40,000 alanine substitutions on the thermal stability of more than 1500 yeast proteins. Using this data, we uncover functionally important residues and protein regions, including those involved in ligand binding and protein-protein interactions. Our work represents the first proteome-wide alanine scan and provides insight into various aspects of protein biology including the structural and functional context underlying mutational sensitivity.
1520
Session 7: Closing Keynote
Chair: Jeff Ranish (ISB)
1525
Ruedi Aebersold (ETHZ)
The adaptable modular proteome specifies cellular states
1600
Closing with Poster Awards
 
1615
Beer and Wine and Tapas Reception
 
1910
Thermo Fisher Scientific Hospitality Night: Seattle Mariners vs. Astros at T-Mobile Park
 

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Executive Committee

Chair: Robert L Moritz
Proteomics Research Laboratory
Institute for Systems Biology, Seattle, WA
Fields of interest: Protein biochemistry, proteomics, mass spectrometry, bioinformatics, chromatography
Vice Chair: Eric W Deutsch
Principal Scientist
Institute for Systems Biology, Seattle, WA
Fields of Interest: Computational proteomics, data standards, PeptideAtlas
Matt Bush
Associate Professor, Department of Chemistry,
University of Washington, Seattle, WA
Fields of interest: Bioanalytical and biophysical chemistry
Andrew Emili
Professor, OHSU Knight Cancer Institute, School of Medicine,
Oregon Health & Science University, Portland, OR
Fields of interest: Functional proteomics, systems biology, protein mass spectrometry
Bill Noble
Professor, Department of Genome Sciences,
University of Washington, Seattle, WA
Fields of interest: statistical and machine learning methods applied to the analysis of complex biological data sets
Chris Overall
Professor, Centre for Blood Research,
University of British Columbia, Vancouver, Canada
Fields of interest: Proteomics, degradomics, Human Proteome Project, proteases, MMPs, extracellular matrix biology, anti-viral immunity, innate immunity
Bhagwat Prasad
Associate Professor, Department of Pharmaceutical Sciences,,
Washington State University, Spokane, WA
Fields of interest: Mechanisms of age, sex, genotype, disease and ethnicity-dependent variability in xeno- and endo-biotic disposition; Interplay of non-CYP enzymes, transporters and microbiome; Physiologically-based pharmacokinetic (PBPK) modeling to predict variability in drug disposition
Dan Raftery
Professor, Dept. of Anesthesiology and Pain Medicine, University of Washington
Director, Northwest Metabolomics Research Center
University of Washington, Seattle WA
Fields of interest: Metabolomics, mass spectrometry, NMR, bioinformatics, cancer metabolism
Martin Sadilek
Mass Spectrometry Facility Manager
University of Washington, Seattle, WA
Fields of interest: Mass spectrometry, metabolomics, lipidomics, instrumentation, fundamentals in analytical chemistry: separation techniques
Judit Villén
Department of Genome Sciences
University of Washington, Seattle, WA
Fields of interest: Proteomics, systems biology, mass spectrometry, cellular signaling, post-translational modifications, protein chemistry