N‐Terminal Sequence Analysis of Proteins and Peptides

Speicher, Kaye D.; Gorman, Nicole; Speicher, David W.

doi:10.1002/0471140864.ps1110s57

Cited by 13 publications

(19 citation statements)

References 13 publications

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“…N-terminal sequencing of NVCP, sucrose gradient centrifugation and electron microscopy for NVCP VLP, and GFP visualization were all performed according to published protocols (Huang, et al 2009; Santi, et al 2008; Speicher, et al 2001). …”

Section: Methodsmentioning

confidence: 99%

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

2011

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Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can established for processing virus-like particles (VLP) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality.

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Section: Methodsmentioning

confidence: 99%

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

2011

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“…Finally, the identified disulfide‐linked peptides are characterized by MS (see Current Protocols article: Zhang, Annan, Carr, & Neubert, , for overview). N‐terminal Edman sequencing (Speicher, Gorman, & Speicher, ) is an older method that was traditionally used to obtain sequence information of reduced peptides. However, this method has largely been replaced by tandem MS (see Current Protocols article: Moore, Young, & Lee, ).…”

Section: Strategic Planningmentioning

confidence: 99%

“…The partial reduction and alkylation strategy was used to determine the three disulfide linkages of the T2 complex. The alkylated cysteine residues are indicated by the italicized lowercase "c." MALDI-MS (Sandoval, 2014) and Edman sequencing (Speicher et al, 2009) of the T2-R3 and T2-R5 complexes revealed disulfide-bond formation between Cys6-Cys36, and between Cys15-Cys25, respectively. Based on the two disulfide bond assignments, the third disulfide bond is deduced to form between resulting in disulfide-cleaved peptides that are further fragmented using CID or HCD for sequence information (Liu, van Breukelen, & Heck, 2014;Ni et al, 2013;Wang, Lu, Wu, Karger, & Hancock, 2011).…”

Section: Figurementioning

confidence: 99%

“…The structure of the labeled peptides is shown in Figure 2. Peptides were identified by MALDI-MS (Sandoval, 2014), and the position of the alkylated cysteines was determined by Edman sequencing (Speicher et al, 2009). The small peak marked with an asterisk showed a molecular mass indicative of T2 with two alkylated cysteines, although the amount of this species was negligible compared with the major species (T2-R3).…”

Section: Of 24mentioning

confidence: 99%

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Experimental Assignment of Disulfide‐Bonds in Purified Proteins

Tang

Speicher

2019

CP Protein Science

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The formation of disulfide bonds in proteins is an important post‐translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified ‐SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence. These assignments provide insights into three‐dimensional structure and contribute to the understanding of structural‐functional relationships. This unit details a series of protocols that have been applied successfully to map disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Mass spectrometry is used to identify disulfide‐containing peptides and determine sites of disulfide linkage. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented. © 2019 by John Wiley & Sons, Inc.

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“…Identification of the N terminal sequence of an intact or cleaved protein is crucial for its biochemical and structural characterization [1]. In conventional shotgun proteomic analysis, it is difficult to identify protein N-termini due to infrequent detection of protein N-terminal peptides.…”

Section: Introductionmentioning

confidence: 99%

Identification of Protein N-Termini Using TMPP or Dimethyl Labeling and Mass Spectrometry

Deng

Zhang

Huang

et al. 2015

Methods in Molecular Biology

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Summary Determination of a protein’s N-terminal sequence can be important for the characterization of protein processing. To increase the confidence of protein N-terminal identification, chemical derivatization of the N-terminal amine group by (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) or dimethyl labeling followed by mass spectrometric analysis is commonly performed. Using this approach, proteins can be separated by SDS-PAGE, and the protein N-terminus of interest is labeled by TMPP or dimethyl in-gel before tryptic digestion and LC-MS analysis. The N-terminus of the protein can thus be easily identified because only the N-terminal tryptic peptide contains the labeling. Peptides with N-terminal derivatization produce a better fragmentation pattern during tandem mass spectrometric analysis, which significantly facilitates sequencing of these peptides.

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N‐Terminal Sequence Analysis of Proteins and Peptides

Cited by 13 publications

References 13 publications

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

Experimental Assignment of Disulfide‐Bonds in Purified Proteins

Identification of Protein N-Termini Using TMPP or Dimethyl Labeling and Mass Spectrometry

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