Sonoreactor-Based Technology for Fast High-Throughput Proteolytic Digestion of Proteins

Rial-Otero, R.; Carreira, Ricardo J.; Cordeiro, Francisco; Moro, Artur J.; Fernandes, Luz; Moura, Isabel; Capelo, José Luís

doi:10.1021/pr060508m

Cited by 38 publications

(30 citation statements)

References 8 publications

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“…18 h), we investigated different digestion procedures to identify the conditions that minimize label loss. Microwave [39, 40], ultrasonic irradiation [41–43] and immobilized enzymes [44] are convenient ways to accelerate protein digestions, so each of these methods was compared to conventional overnight digestion. For each digestion condition, the protein was first labeled with DEPC under conditions known to minimize structural changes to the protein.…”

Section: Resultsmentioning

confidence: 99%

Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Zhou

Vachet

2012

J. Am. Soc. Mass Spectrom.

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Covalent labeling and mass spectrometry are seeing increased used together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g. diethylpyrocarbonate) and non-specific (e.g. hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues, and thus protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g. 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g. microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. As compared to typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 Å to 7 Å for myoglobin, 13 Å to 10 Å for cytochrome c, and 9 Å to 8 Å for β-2-microglobulin.

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Section: Resultsmentioning

confidence: 99%

Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Zhou

Vachet

2012

J. Am. Soc. Mass Spectrom.

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“…Samples selected for analysis were subjected to ultrasonic in-gel enzymatic digestion, according to the ultrafast proteolytic digestion protocol previously described [34,35]. Briefly, protein bands were washed with water and acetonitrile, reduced with DTT and alkylated with IAA in an ultrasonic bath (Sonorex RK 31 H, Bandelin, Berlin, Germany) operating at 35 kHz (100% amplitude).…”

Section: Methodsmentioning

confidence: 99%

Sequential depletion of human serum for the search of osteoarthritis biomarkers

et al. 2012

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BackgroundThe field of biomarker discovery, development and application has been the subject of intense interest and activity, especially with the recent emergence of new technologies, such as proteomics-based approaches. In proteomics, search for biomarkers in biological fluids such as human serum is a challenging issue, mainly due to the high dynamic range of proteins present in these types of samples. Methods for reducing the content of most highly abundant proteins have been developed, including immunodepletion or protein equalization. In this work, we report for the first time the combination of a chemical sequential depletion method based in two protein precipitations with acetonitrile and DTT, with a subsequent two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis for the search of osteoarthritis (OA) biomarkers in human serum. The depletion method proposed is non-expensive, of easy implementation and allows fast sample throughput.ResultsFollowing this workflow, we have compared sample pools of human serum obtained from 20 OA patients and 20 healthy controls. The DIGE study led to the identification of 16 protein forms (corresponding to 14 different proteins) that were significantly (p < 0.05) altered in OA when compared to controls (8 increased and 7 decreased). Immunoblot analyses confirmed for the first time the increase of an isoform of Haptoglobin beta chain (HPT) in sera from OA patients.ConclusionsAltogether, these data demonstrate the utility of the proposed chemical sequential depletion for the analysis of sera in protein biomarker discovery approaches, exhibit the usefulness of quantitative 2D gel-based strategies for the characterization of disease-specific patterns of protein modifications, and also provide a list of OA biomarker candidates for validation.

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“…17 Before protein digestion, the pH of the samples obtained in Section 2.5 was adjusted to 8.0 by adding 1 mL of 0.5 M Ambic. 17 Before protein digestion, the pH of the samples obtained in Section 2.5 was adjusted to 8.0 by adding 1 mL of 0.5 M Ambic.…”

Section: In-solution Protein Digestionmentioning

confidence: 99%

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based profiling as a step forward in the characterization of peritoneal dialysis effluent

et al. 2015

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The aim of this study was to differentiate patients with glomerulonephritis and diabetic nephropathy using (i) peritoneal dialysate effluent, (ii) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and (iii) bioinformatics tools. Profiles of peritoneal dialysate effluent were obtained using (a) sample preparation consisting of protein concentration through centrifugal concentrators and chemical-assisted protein depletion using DL-dithiothreitol, and (b) MALDI-TOF MS. A free open-source bioinformatics tool, Mass-UP, was used to classify such profiles using principal component analysis and hierarchical clustering. The methodology proposed here allows for classifying two different groups of patients with kidney failure, one with chronic glomerulonephritis and other with diabetic nephropathy.

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Sonoreactor-Based Technology for Fast High-Throughput Proteolytic Digestion of Proteins

Cited by 38 publications

References 8 publications

Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

Sequential depletion of human serum for the search of osteoarthritis biomarkers

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based profiling as a step forward in the characterization of peritoneal dialysis effluent

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