2002
DOI: 10.1021/pr025508a
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Integrating “Top-Down” and “Bottom-Up” Mass Spectrometric Approaches for Proteomic Analysis of Shewanella oneidensis

Abstract: Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as… Show more

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Cited by 126 publications
(119 citation statements)
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“…The generated peptides were extracted with 50 % acetonitrile/0.1 % formic acid, and dried before reconstituting in 0.1 % formic acid. The peptides were separated on a C18 column (ResPrep C18 column, Bellefonte, PA, USA) and subjected to nano high performance liquid chromatography (HPLC, Accella, Thermo Fisher) and electro spray ionization (ESI) tandem mass spectrometry (LC-ESI-MS-MS) with a linear trap quadrupole instrument (LTQ, Thermo Fisher Scientific, Waltham, MA) [10][11][12][13]. Tryptic peptides extracted from identical sectors of the same media categories were combined to increase the concentration of the peptides 3-fold prior to mass spectrometry on the LTQ.…”
Section: Proteomic Analysismentioning
confidence: 99%
“…The generated peptides were extracted with 50 % acetonitrile/0.1 % formic acid, and dried before reconstituting in 0.1 % formic acid. The peptides were separated on a C18 column (ResPrep C18 column, Bellefonte, PA, USA) and subjected to nano high performance liquid chromatography (HPLC, Accella, Thermo Fisher) and electro spray ionization (ESI) tandem mass spectrometry (LC-ESI-MS-MS) with a linear trap quadrupole instrument (LTQ, Thermo Fisher Scientific, Waltham, MA) [10][11][12][13]. Tryptic peptides extracted from identical sectors of the same media categories were combined to increase the concentration of the peptides 3-fold prior to mass spectrometry on the LTQ.…”
Section: Proteomic Analysismentioning
confidence: 99%
“…Total peak capacity improvements in multidimensional chromatography platforms have increased the number of detected peptides and proteins identified due to better use of the MS dynamic range and reduced discrimination during ionization. To increase the proteome coverage, particularly for the identification of low-abundance proteins, these peptide-based proteome technologies often require large quantities of enzymatically/chemically cleaved peptides, ranging from a few milligrams [34,35] to several hundreds of micrograms [33,36,37], and are generally incompatible with protein levels extracted from microdissection-procured tissue samples.…”
Section: Capillary Separations For Tissue Proteomics 41 Multidimensimentioning
confidence: 99%
“…Protein separation by gel or liquid chromatography combined with enzymatic cleavage of the intact protein by proteases followed by mass spectrometry (MS) determination of generated peptides and subsequent database searching, has become a popular technique for bottom-up strategy based proteomic study [1][2][3][4][5][6][7]. In this peptide-based technology, rapid and complete digestion of all proteins is crucial to the throughput and accuracy of protein identification.…”
Section: Introductionmentioning
confidence: 99%