Influence of the Protein Staining in the Fast Ultrasonic Sample Treatment for Protein Identification through Peptide Mass Fingerprint and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

Galesio, M.; Vieira, Diana V.; Rial-Otero, R.; Lodeiro, Carlos; Moura, Isabel; Capelo, J.L.

doi:10.1021/pr700850w

Cited by 19 publications

(8 citation statements)

References 13 publications

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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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Section: Methodsmentioning

confidence: 99%

Sequential depletion of human serum for the search of osteoarthritis biomarkers

et al. 2012

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“…Ultrasonic in‐gel enzymatic digestion was done according to the ultrafast proteolytic digestion protocol previously developed in our laboratory 14, 15. Protein bands were manually excised from the gel and placed in safe‐lock tubes of 0.5 mL.…”

Section: Methodsmentioning

confidence: 99%

A comparison of depletion versus equalization for reducing high‐abundance proteins in human serum

et al. 2011

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In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner(™) (PM) have been assessed by 1-D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa.

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“…Then, microwave assisted-extraction (MAE) was evaluated because it has been an interesting alternative compared to conventional solid-liquid extraction methods applied for extracting proteins from plant materials. [22][23][24][25] The interaction of microwave energy with the solvent causes dipole rotation and ionic migration, resulting in heating. This effect of increasing the temperature facilitates the solvent permeation in the cells promoting the disruption of the cell wall and extraction of the target compounds.…”

Section: Total Protein Concentrationmentioning

confidence: 99%

Sample Preparation Applied to Analysis of Proteins in Cowpea Seeds (Vigna unguiculata (L.) Walp.)

Nascimento

Coelho

Júnior

et al. 2018

J. Braz. Chem. Soc.

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In this study, 20 different protocols for protein extraction of cowpea seeds in natura were evaluated taking into account four parameters: cell disruption method, time, temperature and solvent. Factorial design experiments were executed to determine the effects on the total protein content of extracts. The total protein content was determined and the results were in the range of 8.75 ± 1.3 and 86.6 ± 6.9 mg g-1. The richest protein extracts were obtained using Tris-HCl buffer at 50 °C for 20 min for each assessed cell disruption method. However, the results using water were similar to buffer according to t-test at the 95% confidence level. Six protocols demonstrated higher extraction efficiency. Comparative proteomic study using size exclusion chromatography coupled to diode array detector (SEC-DAD) revealed that the one protocol extracted simultaneously high and low molecular mass biomolecules. In fact, the effect of microwave radiation promoted an increased solubility of biomolecules in water. The analysis of chromatographic profiles for principal component analysis (PCA) highlights the greater efficiency of extraction with the application of this protocol. Therefore, our study showed that the optimum condition for extraction of cowpea seed protein consists of water assisted by microwave radiation at 50 °C for 20 min.

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Influence of the Protein Staining in the Fast Ultrasonic Sample Treatment for Protein Identification through Peptide Mass Fingerprint and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

Cited by 19 publications

References 13 publications

Sequential depletion of human serum for the search of osteoarthritis biomarkers

Sequential depletion of human serum for the search of osteoarthritis biomarkers

A comparison of depletion versus equalization for reducing high‐abundance proteins in human serum

Sample Preparation Applied to Analysis of Proteins in Cowpea Seeds (Vigna unguiculata (L.) Walp.)

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