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Comparison of two-dimensional fractionation techniques for shotgun proteomics.

ABSTRACT: Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reversed-phase high-pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reversed-phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high-pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The online pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The offline SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors that contribute to separation efficiency, including resolving power, orthogonality, and sample loss.

SUBMITTER: Dowell JA

PROVIDER: S-EPMC2548321 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Comparison of two-dimensional fractionation techniques for shotgun proteomics.

Dowell James A JA Frost Dustin C DC Zhang Jiang J Li Lingjun L

Analytical chemistry 20080805 17

Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reverse ...[more]

PMID: 18680313

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Project description:To describe the mental architecture between stimulus and response, cognitive models often divide the stimulus-response (SR) interval into stages or modules. Predictions derived from such models are typically tested by focusing on the moment of response emission, through the analysis of response time (RT) distributions. To go beyond the single response event, we recently proposed a method to fractionate verbal RTs into two physiologically defined intervals that are assumed to reflect different processing stages. The analysis of the durations of these intervals can be used to study the interaction between cognitive and motor processing during speech production. Our method is inspired by studies on decision making that used manual responses, in which RTs were fractionated into a premotor time (PMT), assumed to reflect cognitive processing, and a motor time (MT), assumed to reflect motor processing. In these studies, surface EMG activity was recorded from participants' response fingers. EMG onsets, reflecting the initiation of a motor response, were used as the point of fractionation. We adapted this method to speech-production research by measuring verbal responses in combination with EMG activity from facial muscles involved in articulation. However, in contrast to button-press tasks, the complex task of producing speech often resulted in multiple EMG bursts within the SR interval. This observation forced us to decide how to operationalize the point of fractionation: as the first EMG burst after stimulus onset (the stimulus-locked approach), or as the EMG burst that is coupled to the vocal response (the response-locked approach). The point of fractionation has direct consequences on how much of the overall task effect is captured by either interval. Therefore, the purpose of the current paper was to compare both onset-detection procedures in order to make an informed decision about which of the two is preferable. We concluded in favor or the response-locked approach.

Dataset Information

Comparison of two-dimensional fractionation techniques for shotgun proteomics.

Publications

Comparison of two-dimensional fractionation techniques for shotgun proteomics.

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