Proteome analysis ofEscherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

Ihling, Christian; Sinz, Andrea

doi:10.1002/pmic.200401122

Cited by 13 publications

(10 citation statements)

References 39 publications

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“…By combining 2D-DIGE with biochemical prefractionation and the analysis of stationary and exponential growth phases, it was possible to detect and quantify 3199 protein species, among which 575 unique proteins could be identified [4]. In several gel-free approaches using n -dimensional LC for protein [5] or peptide separation [6–9], the number of proteins was successively increased further (Table 1). Most recently, in 2010, Iwasaki and coworkers used 1D-LC/MS/MS with a 350 cm long monolithic silica–C 18 capillary column and 41 h of LC gradient time to identify 2602 proteins [10].…”

Section: Introductionmentioning

confidence: 99%

Optimization of parameters for coverage of low molecular weight proteins

et al. 2010

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Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the analyzer were compared. IT-CID was found to yield the best identification rate, whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4093-x) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Optimization of parameters for coverage of low molecular weight proteins

et al. 2010

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“…Additional information such as isoelectric point, LC elution time or, most commonly, the analysis of peptide fragment ions must be used to distinguish peptides that have identical or very similar masses [11][12][13][14]. Experimental approaches to address this complexity include more extensive protein or peptide separations, or focusing on only those peptides with a specific physical characteristic (e.g., isolation of cysteinyl peptides by chemical labeling or solid phase extraction techniques [15][16][17], and fractionation techniques to add a second separation dimension, e.g., MudPIT [2, 18 -23], in addition to the use of peptide ion fragmentation patterns. While useful, these methods may decrease analysis throughput, result in lower protein coverage, or result in specific protein losses.…”

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confidence: 99%

The Utility of Accurate Mass and LC Elution Time Information in the Analysis of Complex Proteomes

Norbeck

Monroe

Adkins

et al. 2005

J. Am. Soc. Mass Spectrom.

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The combination of mass and normalized elution time (NET) of a peptide identified by liquid chromatography-mass spectrometry (LC-MS) measurements can serve as a unique signature for that peptide. However, the specificity of an LC-MS measurement depends upon the complexity of the proteome (i.e., the number of possible peptides) and the accuracy of the LC-MS measurements. In this work, theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity. Accuracy of the LC-MS measurement of mass-NET pairs (on a 0 to 1.0 NET scale) was described by bivariate normal sampling distributions centered on the peptide signatures. Measurement accuracy (i.e., mass and NET standard deviations of Ϯ0.1, 1, 5, and 10 ppm, and Ϯ0.01 and 0.05, respectively) was varied to evaluate improvements in process quality. The spatially localized confidence score, a conditional probability of peptide uniqueness, formed the basis for the peptide identification. Application of this approach to organisms with comparatively small proteomes, such as Deinococcus radiodurans, shows that modest mass and elution time accuracies are generally adequate for confidently identifying most peptides. For more complex proteomes, more accurate measurements are required. However, the study suggests that the majority of proteins for even the human proteome should be identifiable with reasonable confidence by using LC-MS measurements with mass accuracies within Ϯ1 ppm and high efficiency separations having elution time measurements within Ϯ0.01

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“…The physical manipulations of the PAGE method are very time consuming, and there are inherent limitations such as not providing for the realization of proteins with low and high molecular weight, low and high pI values, and the capture of nonpolar membrane bound proteins. 3,4 Recently, alternative methods have been developed for direct protein processing with liquid and/or stationary supports. 3,[5][6][7]9,10 Preprocessing consisted of protein precipitation, denaturation using concentrated urea to remove the protein secondary and tertiary structures, dithiothreitol (DTT) disulfide reduction, and alkylation steps.…”

Section: Introductionmentioning

confidence: 99%

“…An important method of protein separation that has found extensive utility is two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE). ,, However, processing of the many separated proteins is performed by excising a protein spot from the gel with subsequent purification, concentration, and trypsin digestion. The physical manipulations of the PAGE method are very time consuming, and there are inherent limitations such as not providing for the realization of proteins with low and high molecular weight, low and high pI values, and the capture of nonpolar membrane bound proteins. , …”

Section: Introductionmentioning

confidence: 99%

A Protein Processing Filter Method for Bacterial Identification by Mass Spectrometry-Based Proteomics

et al. 2010

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A "one-pot" alternative method for processing proteins and isolating peptide mixtures from bacterial samples is presented for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data reduction. The conventional in-solution digestion of the protein contents of bacteria is compared to a small disposable filter unit placed inside a centrifuge vial for processing and digestion of bacterial proteins. Each processing stage allows filtration of excess reactants and unwanted byproduct while retaining the proteins. Upon addition of trypsin, the peptide mixture solution is passed through the filter while retaining the trypsin enzyme. The peptide mixture is then analyzed by LC-MS/MS with an in-house BACid algorithm for a comparison of the experimental unique peptides to a constructed proteome database of bacterial genus, specie, and strain entries. The concentration of bacteria was varied from 10 × 10(7) to 3.3 × 10(3) cfu/mL for analysis of the effect of concentration on the ability of the sample processing, LC-MS/MS, and data analysis methods to identify bacteria. The protein processing method and dilution procedure result in reliable identification of pure suspensions and mixtures at high and low bacterial concentrations.

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Proteome analysis ofEscherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

Cited by 13 publications

References 39 publications

Optimization of parameters for coverage of low molecular weight proteins

Optimization of parameters for coverage of low molecular weight proteins

The Utility of Accurate Mass and LC Elution Time Information in the Analysis of Complex Proteomes

A Protein Processing Filter Method for Bacterial Identification by Mass Spectrometry-Based Proteomics

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