Kevin M Buck’s research while affiliated with University of Wisconsin–Madison and other places

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Publications (10)


Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate
  • Article

February 2024

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20 Reads

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6 Citations

Journal of the American Society for Mass Spectrometry

Emily A Chapman

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Brad H Li

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[...]

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High-throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF
  • Preprint
  • File available

August 2023

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35 Reads

The extracellular matrix (ECM) is a complex assembly of proteins that provide interstitial scaffolding and elastic recoil to human lungs. The pulmonary extracellular matrix (ECM) is increasingly recognized as an independent bioactive entity by creating biochemical and mechanical signals that influence disease pathogenesis, making it an attractive therapeutic target. However, the pulmonary ECM proteome (matrisome) remains challenging to analyze by mass spectrometry due to its inherent biophysical properties and relatively low abundance. Here, we introduce a strategy designed for rapid and efficient characterization of the human pulmonary ECM using the photocleavable surfactant Azo. We coupled this approach with trapped ion mobility MS with diaPASEF to maximize depth of matrisome coverage. Using this strategy, we identify nearly 400 unique matrisome proteins with excellent reproducibility that are known to be important in lung biology, including key insoluble ECM proteins.

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Figure 2. Sarcopenia is associated with changes in muscle composition. (A) Schematic showing the age of rhesus monkeys used in this study relative to a generalized survival curve. (B) Body weight and body mass index (BMI) for young (red) middle-age (purple), and old (blue) monkeys (n=4 per group). (C) Electrical properties of the upper leg (determined by S-BIS), extracellular to intracellular water ratios, phase angle, and reactance curves. (D) Distribution of fiber types vastus laterals biopsies detected using antibodies specific to type I or type II myosin heavy chain. (E) Quantification of non-contractile content (black arrows) in hematoxylin and eosin stained sections (left) and fibrotic content (right) as detected by picrosirius expressed as percent area of tissue section (4-5 images per animal). Data shown as median +/-IQR, #p<0.1, * p>0.05, ** p<0.01.
Figure 4. Gene Ontology (GO) term enrichment analysis of altered proteins from aging rhesus monkey skeletal muscle. Significantly altered proteins (Table S1) were subject to a GO term analysis to identify associated biological processes (Table S2). Shown are relative peak intensities for selected proteins related to selected biological functions for young, middle, and old rhesus monkeys (n=4 per group): (C) ATP metabolic process (GO: 0046034), (D) AMP metabolic process (GO: 0046033), and (E) fatty acid beta oxidation (GO:0006635). *p<0.01, ** p<0.001, ***p<0.0001, and ****p<0.0001. ITA6=integrin alpha-6, ITA7= integrin alpha-6, COL6A1=collagen alpha-1 (VI) chain, COL6A3=collagen alpha-3 (VI) chain, MYL9=myosin regulatory light polypeptide 9, MYH11=myosin-11, ACTN1=alpha-actinin-1, ACTN4=, ATPD = ATP synthase subunit d, mitochondrial, DHSD=succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial, QCR8 = cytochrome b-c1 complex subunit 8 (QCR8), AMPD= AMP deaminase 1, PUR8 = adenylosuccinate, PURA1 = adenylosuccinate synthetase isozyme 1, ECI1 = enoylCoA delta isomerase 1, mitochondrial, THIM = acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha 2, and ECHD1=ethylmalonyl-CoA decarboxylase.
Mass spectrometry-based multi-omics identifies metabolic signatures of sarcopenia in rhesus monkey skeletal muscle

July 2023

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90 Reads

Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in omics technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified numerous proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified multiple metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offer new insights into the molecular mechanism underlying sarcopenia for the evaluation and monitoring of therapeutic treatment of sarcopenia.


One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant

May 2022

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27 Reads

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14 Citations

Analytical Chemistry

Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with extremely high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content in exosomes, but the current methods require laborious and time-consuming multistep sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, to simplify exosomal lysis, effectively extract proteins, and expedite digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3466 proteins and quantified 2288 proteins using a reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. Here, 3166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision that this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.


One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant

March 2022

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20 Reads

Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content of exosomes but the current methods require laborious and time-consuming multi-step sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, for rapid and effective exosomal lysis, protein extraction, and digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3,466 proteins and quantified 2,288 proteins using reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. 3,166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.


High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry

July 2021

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169 Reads

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40 Citations

Analytical Chemistry


Figure 2. Azo promotes rapid and reproducible tryptic digestion for quantitative proteomics. (A) SDS-PAGE showing tryptic digestion efficiency in 0.1% Azo after 0.5 and 24 h of digestion. (B,C) Venn diagrams illustrating overlap in unique protein groups (B), and unique peptides (C) identified by MaxQuant between 30 min, 1 h, and 24 h tryptic digestions (n = 3 technical replicates for each group). (D-F) Scatterplots of Log 2 LFQ protein intensities showing high reproducibility between replicates from 30 min (D), 1 h (E), and 24 h (F) tryptic digestions. Pearson correlation coefficients are shown in the top left corner of each panel. (G-I) Scatterplots of Log 2 LFQ protein intensities showing high reproducibility between averaged replicates from 30 min digestions plotted against 1 h digestions (G), 30 min digestions plotted against 24 h digestions (H), and 1 h digestions plotted against 24 h digestions (I) with Pearson correlation coefficients shown in the top left corner of each panel (n = 3 technical replicates for each group).
Figure 3. Reproducible protein identification and quantitation from small amounts of tissue. (A,B) Venn diagrams illustrating overlap in unique protein groups (A), and unique peptides (B) identified by MaxQuant between 20, 5, and 1mg of tissue (n = 3 technical replicates for each group). (D Scatterplots of Log2 L FQ protein intensities showing high reproducibility between replicates from 20 mg (D), 5 mg (E), and 1 mg (F) tissue extractions. Pearson correlation coefficients are shown in the top left corner of each panel. (G-I) Scatterplots of Log2 L FQ protein intensities showing high reproducibility between averaged replicates from 1 mg extractions plotted against 5 mg extractions (G), 1 mg extractions plotted against 20
Figure 4. LFQ analysis with high reproducibility from low sample loading amounts on the timsTOF Pro. (A,B) Bar charts showing the total number of unique protein groups (A), and unique peptides (B), identified by MaxQuant from injections of 200, 100, 50, 25, 12.5, and 6.25 ng of digested peptide (n = 3 technical replicates for each group), respectively. Total number of identifications are displayed above each bar. (C,D) Scatterplots of Log 2 LFQ protein intensities showing high reproducibility between replicates from 200 ng (C) and 6.25 ng (D) injections. Pearson correlation coefficients are shown in the top left corner of each panel.
Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro

July 2021

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182 Reads

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44 Citations

Journal of Proteome Research

Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample cleanup to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.


Figure 4. LFQ analysis with high reproducibility from low sample loading amounts on the timsTOF Pro. (A and B). Bar charts showing the total number of unique protein groups (A), and unique peptides (B), identified by MaxQuant from injections of 200, 100, 50, 25, 12.5, and 6.25 ng of digested peptide (n=3 for each group), respectively. Total number of identifications are displayed above each bar. (C and D). Scatterplots of Log2 LFQ protein intensities showing high reproducibility between replicates from 200 ng (C) and 6.25 ng (D) injections. Pearson correlation coefficients are shown in the top left corner of each panel.
Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro

May 2021

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110 Reads

Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample clean-up to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4,000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.

Citations (5)


... Proteins were extracted from cardiac left ventricular tissue as previously reported. 18,19 No reducing agents were added to the extraction buffers to preserve protein SSG. Following the extraction procedure, the protein-enriched cardiac tissue lysate was passed through a Titan3, 17mm PES membrane syringe filter (pre-soaked with LiCl extraction buffer) using a 5 mL Luer-lock syringe into an Eppendorf Protein Lo-Bind tube, snap-frozen in liquid nitrogen, and stored at -80 ºC until topdown proteomics analysis. ...

Reference:

In-depth Characterization of S-Glutathionylation in Ventricular Myosin Light Chain 1 Across Species by Top-Down Proteomics
Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate
  • Citing Article
  • February 2024

Journal of the American Society for Mass Spectrometry

... [21][22][23] Various organisms have been used to establish muscle atrophy models, including rats, mice, Drosophila, Caenorhabditis elegans, zebrafish, African turquoise killifish, medaka, rhesus monkeys, rabbits, Yucatan minipigs, and so forth. [24][25][26][27][28] These models are induced using different methods and are instrumental in studying sarcopenia and cachexia ( Figure 1). In sarcopenia research, the natural aging model is the most commonly used, as it accurately reflects the physiological changes observed in age-related muscle atrophy in humans. ...

Mass Spectrometry-Based Multiomics Identifies Metabolic Signatures of Sarcopenia in Rhesus Monkey Skeletal Muscle
  • Citing Article
  • November 2023

Journal of Proteome Research

... First studies show promising results, for example, PASEF was first successfully employed to aid in the identification of more than 6000 protein groups in a 200 ng single-run for HeLa standard digest (Meier et al., 2018). Specifically, in case of small extracellular vesicles, one study used the PASEF method to identify 3466 unique protein groups in a triplicate run of cell-derived sEV (Buck et al., 2022). Serum-derived vesicles were characterized using this technique with 915 protein identifications in a study comparing samples from healthy donors and patients suffering from dermatomyositis or polymyositis (Meng et al., 2023). ...

One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant
  • Citing Article
  • May 2022

Analytical Chemistry

... 8 In conjunction with collision-induced unfolding (CIU), IM/MS has been applied to probe structures and stabilities of multi-domain proteins, including mAbs. [9][10][11][12] In combination with molecular dynamics (MD) simulations, IM/MS suggested that the broad peaks in IM/MS spectra of mAbs may be related to their hinge motions. 13 Nevertheless, conventional IM/MS methods conduct ensemble-type measurements that characterise the entirety of the mAb conformational space and hence characterise their structures or stabilities only by their ensemble averages. ...

High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry

Analytical Chemistry

... The sensitivity of mass spectrometers has dramatically improved in recent years due to, for example, increased ionization efficiency, improved and enhanced ion transfer, and advances in DDA (data-dependent analysis) due to the introduction of new fragmentation approaches such as PASEF (parallel serial fragmentation) combined with ion mobility separation and fast scanning TOF (time-of-flight) detector [44,56,[66][67][68][69][70][71]. ...

Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro

Journal of Proteome Research