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Advancements in life science research are driven by innovations in technology for genomics and proteomics applications. Gel electrophoresis, a powerful tool used in the analysis of a variety of biologically interesting molecules (e.g., nucleic acids and proteins), continues to be the method of choice for the separation and visualization of biomolecules.
For proteins, gel separations can provide a clear picture of a number of characteristics of the sample, including its complexity (number and relative abundance of components), state (apparent sample integrity), purity (level of salts and other impurities), and physical properties inherent to the molecule (molecular weight and isoelectric point).
Subsequent preparations and analyses of the proteins present in the gel can provide additional information about the original sample. Mass spectrometry is often the method of choice for analyzing the samples. Prior to analysis, the proteins may be digested “in-gel,” with the resultant peptides extracted and analyzed for protein identification in a bottom-up approach. Alternatively, many investigators prefer to analyze the intact proteins directly for the determination of highly accurate molecular weights, and for structural characterization after fragmentation in the mass spectrometer [e.g., by electron capture dissociation (ECD)]. This type of top-down approach for gel-separated proteins is made challenging by the inefficiency of traditional approaches (passive elution and gel electroelution) for extraction of the intact proteins from the gel matrix and the difficulties presented by the impurities in the sample, the lack of reproducible performance, and low throughput.
Utilizing advances in microfluidic technology, a new instrument, the Gel Protein Recovery system (GPR-800), has been developed to revolutionize the extraction of biomolecules, including proteins, peptides, and nucleic acids, from polyacrylamide and agarose gels. Proprietary developments in controlling electrokinetic flow have led to the development of a novel method for coupling high efficiency and robust electroelution with sample recovery decoupled from the electric field. The GPR-800 instrument delivers 500 V per channel for rapid sample extraction, typically ≤15 min. Each GPRchip contains an array of channels for processing up to eight samples simultaneously. The combination of high-voltage electroelution and the 8-plex GPRchip permit highly efficient sample recoveries with excellent throughput.
Improvements in the purity of the samples post-extraction have been accomplished through buffer optimization featuring the Progenta family of acid labile surfactants. Advances in synthetic chemistry have led to “smart surfactants” that can be used for sample preparation and processing, and then be rapidly broken down by decreasing the pH of the sample solution. The degradation by-products do not possess surfactant properties or interfere with analysis by mass spectrometry. The combination of the efficient electroelution with the optimized reagents leads to recovery of high-quality samples for reliable mass spectrometry data.
ExperimentalStandard protein samples were separated on 10% SDS-PAGE gels and visualized by Coomassie staining. 2-mm round protein gel spot samples were cut out and placed into primed Protein GPRchips. The prepped chips were placed in the GPR-800 system and electroelution was performed for 15 min at 500 V.
Following extraction of the intact proteins from the gel spots, the recovered samples were transferred to microcentrifuge tubes. Surfactant degradation was performed by the addition of trifluoroacetic acid (TFA) or formic acid–based reagents for subsequent analysis by MALDI and ESI mass spectrometry, respectively. The samples for MALDI analysis were lyophilized, reconstituted in a reversed phase loading buffer, spotted with C4 LithTips using CHCA matrix, and analyzed on an ABI 4800 TOF/TOF mass spectrometer in the linear detection mode. The samples for ESI analysis were lyophilized, reconstituted in a reversed phase loading buffer, loaded onto a Zorbax C4 column, and analyzed by LC-MS on a Thermo LTQ-XL mass spectrometer.
ResultsStandard proteins from a variety of molecular weights (6–65 kDa) and initial gel loads (0.1–2.0 µg) were extracted with the Gel Protein Recovery system (GPR-800) and analyzed by MALDI and ESI mass spectrometry.
Figure 1 shows representative MALDI mass spectra from small proteins (aprotinin, lysozyme, and myoglobin) recovered from SDS-PAGE gels containing 1.0 µg gel loads. Intense intact protein peaks are observed for each protein: aprotinin (6495.4 Da), lysozyme (14294.3 Da), and myoglobin (16997.0 Da). Figure 2 shows representative MALDI mass spectra from medium-sized proteins (carbonic anhydrase and BSA) recovered from 2.0 µg gel loads using the GPR-800. Intact protein peaks (+1 charge state) can be seen for both proteins (29037 and 66863 Da, respectively), as well as a +2 peak for BSA.
In Figure 3, a 1.0-µg myoglobin sample was extracted with the GPR-800 for analysis by LC-MS on an electrospray ionization mass spectrometer. The resultant charge state envelope of myoglobin (A) was deconvoluted to obtain the molecular weight of the protein (B) as 16992.8 Da.
Conclusions
The Gel Protein Recovery system (GPR-800) can be effectively utilized to recover intact proteins from polyacrylamide gels. The extracted protein samples can be prepped for direct analysis by mass spectrometry for intact mass measurements and/or top-down characterization. The mass spectra shown in Figures (123) demonstrate the sample quality of the proteins after GPR processing. The proprietary decoupled sample recovery process used in the GPRchips permits reproducible recovery over a range of molecular weights (6–65 kDa) and initial gel loads (0.1–2.0 µg). Coupling these developments in electrokinetic transport with advancements in surfactant chemistry the GPR-800 system is a versatile tool for the recovery of biomolecules from acrylamide and agarose gels.
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