Microwave assisted acid cleavage for denaturation and proteolysis of intact human adenovirus

Abstract

Microwave assisted acid cleavage was applied directly to intact adenovirus type 5 to achieve denaturation and proteolysis in a single reaction. The speed of the digestion, coupled with the simplicity of MALDI analysis, allowed peptide products to be profiled in less than 5 min. Identification of peptides from a range of proteins by MALDI-TOF confirms that both denaturation and proteolysis were achieved using low concentrations of acetic acid (12.5%) and short incubations (1.5 to 2 min) at high temperatures (140° C). These conditions favor production of peptides that carry Asp on their C-termini. When this cleavage reaction is carried out in highly enriched H 2 18 O, a single atom of 18 O is introduced site-specifically into the carboxyl terminal. The labeling reaction is applied to label reporter peptides from human adenovirus type 5 harvested from HeLa cells. Small peptide products of endogenous processing were also observed. Adenoviruses are commonly used as vectors in genetic engineering and gene therapy [1] and, as such, have generated continuing interest in new methods for their rapid characterization [for example, 2-4] The adenovirus particle is a large macromolecular assembly (M r ~ 2×10 8 Da) composed of a double-stranded DNA genome and multiple copies of at least 13 distinct proteins [3,[5][6][7][8][9][10][11]. A viral protease is responsible for processing [12][13][14]. The small proteome of the adenovirus served earlier as a model system for the development in this laboratory of enzyme-catalyzed 18 O-labeling [15]. In the present report adenovirus type 5 is revisited to demonstrate the capability of Asp-selective rapid microwave-assisted acid cleavage [16][17][18][19][20][21][22] Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access 1.3Figure 1 illustrates the dominant mechanism by which acid cleavage is thought to occur [28] in a protein, and the pathway by which it is expected that a single atom of 18 O would be incorporated into proteolytic products. 18 O-labeling was first optimized on myoglobin and ovalbumin, and then extended to label peptides from human adenovirus type 5.The effect of acid type and concentration on the extent of incorporation of the isotope label has been evaluated. The site of incorporation is confirmed using laser induced dissociation. Material and methods 2.1Human adenovirus type 5 (ATCC # VR-5) was cultured in HeLa host cells (ATCC # CCL-2) following a published method [15]. HeLa cells were grown in 150 cm 2 culture flasks to about 90% confluence, and exposed to virus stock for 1.5 hr with slow rocking. The viru...

The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein

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Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

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Rapid analysis of ricin using hot acid digestion and MALDI‐TOF mass spectrometry

“…Acid‐catalyzed proteolysis has been shown to cleave with high specificity on the carboxyl and amino bonds of aspartic acid residues in proteins . After microwave‐assisted hot acid digestion of 100 ng commercial ricin, 44 peptide peaks were observed in the mass range 1000 to 5000 Da with signal to noise ratios >10 (Figure and Supplementary Table 1).…”

“…To address current limitations in mass spectrometry‐based ricin detection methods, a novel strategy is reported here to characterize ricin using hot acid digestion. Microwave‐assisted hydrolysis provides residue‐specific protein cleavage, and peptide products are characterized by MALDI‐TOF mass spectrometry. Post source decay (PSD) analysis in the reflectron mode coupled with online protein database searching was used to increase tolerance to contamination and the specificity of the analysis.…”