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Centrifugal methods and devices for rapid in-gel digestion of proteins.

ABSTRACT: Modern proteomic research frequently relies upon separation of proteins in a polyacrylamide gel matrix followed by in-gel enzymatic digestion and extraction of peptides for subsequent analysis by MS. In this work, we propose a novel semi-automated method of mechanical processing of gel bands by passing these bands through a specially designed centrifugal device termed a Gel Shredder prior to digestion and extraction of peptides. Such a device allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, approximately 150-300 microm, prior to the enzymatic digestion and extraction of peptides. Shredding into uniform blocks increases the surface area of the gel pieces and promotes improved gel rehydration, allowing improved diffusion of the proteolytic enzymes and solvent into the gel lattice. We demonstrate that the new method substantially reduces the time spent on tedious manual handling of gel bands, while minimizing the risk of sample contamination. The performance of the Gel Shredder has been compared with a conventional in-gel digestion protocol using several standard proteins and a complex proteomic sample in terms of relative quantitation by either MALDI-TOF/TOF or nanoLC-ESI IT-Fourier transformation ion cyclotron resonance MS. It is shown that significant time savings and improved peptide recovery can be obtained for many proteins using the Gel Shredder compared with the traditional in-gel digestion protocol.

SUBMITTER: Lazarev AV

PROVIDER: S-EPMC2907145 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Centrifugal methods and devices for rapid in-gel digestion of proteins.

Lazarev Alexander V AV Rejtar Tomas T Dai Shujia S Karger Barry L BL

Electrophoresis 20090301 6

Modern proteomic research frequently relies upon separation of proteins in a polyacrylamide gel matrix followed by in-gel enzymatic digestion and extraction of peptides for subsequent analysis by MS. In this work, we propose a novel semi-automated method of mechanical processing of gel bands by passing these bands through a specially designed centrifugal device termed a Gel Shredder prior to digestion and extraction of peptides. Such a device allows integrated washing, destaining and shredding o ...[more]

PMID: 19309014

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Project description:BackgroundThe success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples.MethodsRecovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated.ResultsApplying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K.ConclusionsAmicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used.

Project description:BACKGROUND:The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins. Elimination of highly enriched lipids and interfering substances from raft samples is generally required before mass spectrometric analyses can be performed, but these procedures often lead to excessive protein loss and increased sample variability. For accurate analyses of the raft proteome, simplified protocols are needed to avoid excessive sample handling and purification steps. RESULTS:We have devised a simple protocol using a 'tube-gel' protein digestion that, when combined with mass spectrometry, can be used to obtain comprehensive and reproducible identification and quantitation of the lipid raft proteome prepared from neonatal mouse brain. Lipid rafts (detergent-resistant membranes using Triton X-100 extraction) prepared from neonatal mouse brain were directly incorporated into a polyacrylamide tube-gel matrix without prior protein separation. After in-gel digestion of proteins, nanospray LC-MS/MS was used to analyze the extracted peptides, and the resulting spectra were searched to identify the proteins present in the sample. Using the standard 'label-free' proteomics approach, the total number of MS/MS spectra for the identified proteins was used to provide a measure of relative protein abundances. This approach was successfully applied to lipid rafts prepared from neonatal mouse brain. A total of 216 proteins were identified: 127 proteins (58.8%) were predicted to be membrane proteins, or membrane-associated proteins and 175 proteins (~80%) showed less than a 2-fold variation in the relative abundance in replicate samples. CONCLUSION:The tube-gel protein digestion protocol coupled with nanospray LC-MS/MS (TubeGeLC-MS/MS) offers a simple and reproducible method for identifying and quantifying the changes of relative abundances in lipid raft proteins from neonatal mouse brain and could become a useful approach for studying lipid raft proteins from various tissues.

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Centrifugal methods and devices for rapid in-gel digestion of proteins.

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Centrifugal methods and devices for rapid in-gel digestion of proteins.

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