Simultaneous Quantification of Protein Expression and Modifications by Top-down Targeted Proteomics: A Case of the Sarcomeric Subproteome

Lin, Ziqing; Wei, Liming; Cai, Wenxuan; Zhu, Yanlong; Tucholski, Trisha; Mitchell, Stanford D.; Guo, Wei; Ford, Stephen P.; Diffee, Gary M.; Ge, Ying

doi:10.1074/mcp.tir118.001086

Cited by 28 publications

(32 citation statements)

References 72 publications

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“…Top-down mass spectrometry (MS)-based proteomics is the premier tool to comprehensively characterize proteoforms, the myriad of protein products arising from a single gene due to genetic variants, alternative RNA splicing, and PTMs. − In particular, top-down tandem MS (MS/MS) can characterize proteoforms of interests and locate sites of PTMs and amino acid sequence variations. − Likewise, top-down proteomics can effectively distinguish isoforms encoded by different genes from a gene family, which often exhibit high sequence homology (i.e., α-Tpm and β-Tpm are isoforms that come from two different genes, Tpm1 and Tpm2 , respectively). ,− We have recently established a top-down liquid chromatography (LC)–MS/MS method that can rapidly analyze skeletal muscle tissues and its application to sarcopenia, an age-related loss of skeletal muscle mass and function. ,, However, a study to comprehensively characterize the heterogeneity of myofilament proteoforms in a large number of muscles representing a range of functional and biochemical properties is lacking.…”

Section: Introductionmentioning

confidence: 99%

Top-Down Proteomics Reveals Myofilament Proteoform Heterogeneity among Various Rat Skeletal Muscle Tissues

et al. 2019

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Heterogeneity in skeletal muscle contraction time, peak power output, and resistance to fatigue, among others, is necessary to accommodate the wide range of functional demands imposed on the body. Underlying this functional heterogeneity are a myriad of differences in the myofilament protein isoform expression and post-translational modifications; yet, characterizing this heterogeneity remains challenging. Herein, we have utilized top-down liquid chromatography (LC)-mass spectrometry (MS)-based proteomics to characterize myofilament proteoform heterogeneity in seven rat skeletal muscle tissues including vastus lateralis, vastus medialis, vastus intermelius, rectus femoris, soleus, gastrocnemius, and plantaris. Top-down proteomics revealed myofilament proteoforms varied greatly across the seven different rat skeletal muscle tissues. Subsequently, we quantified and characterized myofilament proteoforms using online LC-MS. We have comprehensively characterized the fast and slow skeletal troponin I isoforms, which demonstrates the ability of top-down MS to decipher isoforms with high sequence homology. Taken together, we have shown that top-down proteomics can be used as a robust and highthroughput method to characterize the molecular heterogeneity of myofilament proteoforms from various skeletal muscle tissues.

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Section: Introductionmentioning

confidence: 99%

Top-Down Proteomics Reveals Myofilament Proteoform Heterogeneity among Various Rat Skeletal Muscle Tissues

et al. 2019

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“…3 and Supplementary Figs. 21 and 22 shown were smoothened by Gauss algorithm with a smoothing width of 1.67 s. To quantify protein expression across samples, the Top 5 most abundant charge states' ions (average ± 0.2 m/z) of all major proteoforms from the same protein were retrieved collectively as one extracted peak in the EIC 61 . The area under curve was manually determined for each protein isoform using DataAnalysis.…”

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Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum

et al. 2020

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Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from blood remains an unsolved challenge due to the extraordinary dynamic range of the blood proteome. Here, we develop an integrated nanoproteomics method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of cardiac troponin I (cTnI), a gold-standard cardiac biomarker, directly from serum. These NPs enable the sensitive enrichment of cTnI (<1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant proteins such as human serum albumin (>10 10 more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide highresolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibodyfree strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.

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“…This limitation is common to other top-down MS methods. In fact, although the more commonly used ESI-based top-down approaches typically provide enough sequence information for identification, localization, and quantification of a few positional isomers of modified proteins, [54][55][56] the analysis of complex mixtures of isomers is more difficult. 57 The sensitivity of bottom-up MS analysis was improved by selected ion monitoring.…”

Section: Structural Characterization Of A2v Bsab By Top-down and Middle-down Maldi-isd Ft-icr Msmentioning

confidence: 99%

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry

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et al. 2019

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Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The “bi-specificity” of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd’ regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.

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Simultaneous Quantification of Protein Expression and Modifications by Top-down Targeted Proteomics: A Case of the Sarcomeric Subproteome

Cited by 28 publications

References 72 publications

Top-Down Proteomics Reveals Myofilament Proteoform Heterogeneity among Various Rat Skeletal Muscle Tissues

Top-Down Proteomics Reveals Myofilament Proteoform Heterogeneity among Various Rat Skeletal Muscle Tissues

Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry

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