Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

Cai, Wenxuan; Tucholski, Trisha; Chen, Bifan; Alpert, Andrew J.; McIlwain, Sean J.; Kohmoto, Takushi; Jin, Song; Ge, Ying

doi:10.1021/acs.analchem.7b00380

Cited by 110 publications

(173 citation statements)

References 48 publications

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“…top-down, middle-down, and bottom-up proteomics. 6–11 For bottom-up proteomics, proteins are digested enzymatically and/or chemically into peptides. The resulting peptides are separated by high-performance liquid chromatography (HPLC), and the eluted peptides are subsequently subjected to mass spectrometric (MS) analysis.…”

Section: Introductionmentioning

confidence: 99%

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

et al. 2017

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Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, the reaction incompletion and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with the commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, Thiopropyl-Sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.

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Section: Introductionmentioning

confidence: 99%

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

et al. 2017

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“…From a technical perspective, there is a preference for identification of proteoforms <25 kDa using the Q Exactive MS and fragmentation is limited to higher energy collision dissociation (HCD); however, these limitations can be overcome with more advanced instrumentation 78–79 . Moreover, depth of coverage may be increased if a deglycosylation step prior to MS were performed, as many secreted proteins are N-glycosylated and this post-translational modification can complicate top down MS analysis.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin

et al. 2018

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Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, proteins, and oligosaccharides. This study presents a first step towards identifying soluble mediators of keratinocyte-sensory neuron communication by evaluating the potential for top down mass spectrometry to identify proteoforms released during one minute of mechanical stimulation of mouse skin from naïve animals. Overall, this study identified 47 proteoforms in the secretome of mouse hind paw skin, of which 14 were differentially released during mechanical stimulation, and includes proteins with known and previously unknown relevance to mechanotransduction. Finally, this study outlines a bioinformatic workflow that merges output from two complementary analysis platforms for top down data and demonstrates the utility of this workflow for integrating quantitative and qualitative data.

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“…Ge and co‐workers recently introduced serial size exclusion chromatography (sSEC), which combines multiple matrix pore sizes for separation of complex protein mixtures in MS‐compatible buffers . SEC is a non‐adsorptive mode of liquid chromatography that sorts molecules based on their size and differential access to matrix pore volume .…”

Section: Separation Of Proteoformsmentioning

confidence: 99%

Identification and Quantification of Proteoforms by Mass Spectrometry

et al. 2019

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A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.

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Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy

Cited by 110 publications

References 48 publications

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin

Identification and Quantification of Proteoforms by Mass Spectrometry

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