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Chromatography, Volume 1, Issue 4 (December 2014), Pages 159-226

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Open AccessArticle Electrophoretic Extraction and Proteomic Characterization of Proteins Buried in Marine Sediments
Chromatography 2014, 1(4), 176-193; doi:10.3390/chromatography1040176
Received: 1 July 2014 / Revised: 11 September 2014 / Accepted: 18 September 2014 / Published: 13 October 2014
Cited by 3 | PDF Full-text (646 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Proteins are the largest defined molecular component of marine organic nitrogen, and hydrolysable amino acids, the building blocks of proteins, are important components of particulate nitrogen in marine sediments. In oceanic systems, the largest contributors are phytoplankton proteins, which have been tracked from
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Proteins are the largest defined molecular component of marine organic nitrogen, and hydrolysable amino acids, the building blocks of proteins, are important components of particulate nitrogen in marine sediments. In oceanic systems, the largest contributors are phytoplankton proteins, which have been tracked from newly produced bloom material through the water column to surface sediments in the Bering Sea, but it is not known if proteins buried deeper in sediment systems can be identified with confidence. Electrophoretic gel protein extraction methods followed by proteomic mass spectrometry and database searching were used as the methodology to identify buried phytoplankton proteins in sediments from the 8–10 cm section of a Bering Sea sediment core. More peptides and proteins were identified using an SDS-PAGE tube gel than a standard 1D flat gel or digesting the sediment directly with trypsin. The majority of proteins identified correlated to the marine diatom, Thalassiosira pseudonana, rather than bacterial protein sequences, indicating an algal source not only dominates the input, but also the preserved protein fraction. Abundant RuBisCO and fucoxanthin chlorophyll a/c binding proteins were identified, supporting algal sources of these proteins and reinforcing the proposed mechanisms that might protect proteins for long time periods. Some preserved peptides were identified in unexpected gel molecular weight ranges, indicating that some structural changes or charge alteration influenced the mobility of these products during electrophoresis isolation. Identifying buried photosystem proteins suggests that algal particulate matter is a significant fraction of the preserved organic carbon and nitrogen pools in marine sediments. Full article
Open AccessArticle Description of the Retention and Peak Profile for Chromolith Columns in Isocratic and Gradient Elution Using Mobile Phase Composition and Flow Rate as Factors
Chromatography 2014, 1(4), 194-210; doi:10.3390/chromatography1040194
Received: 22 October 2014 / Revised: 13 November 2014 / Accepted: 14 November 2014 / Published: 19 November 2014
Cited by 3 | PDF Full-text (895 KB) | HTML Full-text | XML Full-text
Abstract
The effect of the modifier concentration and flow rate on the chromatographic performance of a second generation Chromolith® RP-18e column, under isocratic and gradient elution with acetonitrile-water mixtures, was examined using four sulphonamides as probe compounds. The acetonitrile concentration was varied between 5
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The effect of the modifier concentration and flow rate on the chromatographic performance of a second generation Chromolith® RP-18e column, under isocratic and gradient elution with acetonitrile-water mixtures, was examined using four sulphonamides as probe compounds. The acetonitrile concentration was varied between 5 and 55% (v/v), and the flow rate between 0.1 and 5.0 mL/min, keeping the other factors constant. The changes in both retention and peak profile were modelled, and used to build simple plots, where the logarithm of the retention factor was represented against the modifier concentration (in gradient elution, against the initial modifier concentration), and the half-widths or widths against the retention time (in gradient elution, against the time at the column outlet). A particular plot was needed for describing the retention of each sulphonamide, but due to the similar interaction kinetics, a unique plot described the changes in the half-widths for all four sulphonamides. The changes in retention with the flow showed that allegedly in the second generation Chromolith, the column deformation observed for the first generation Chromolith, with the applied pressure at increasing flow, is decreased. Full article
(This article belongs to the Special Issue Monolithic Columns in Separation Sciences)
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Open AccessArticle Analyses of Phytohormones in Coconut (Cocos Nucifera L.) Water Using Capillary Electrophoresis-Tandem Mass Spectrometry
Chromatography 2014, 1(4), 211-226; doi:10.3390/chromatography1040211
Received: 8 October 2014 / Revised: 8 December 2014 / Accepted: 12 December 2014 / Published: 22 December 2014
Cited by 2 | PDF Full-text (1228 KB) | HTML Full-text | XML Full-text
Abstract
Capillary electrophoresis (CE) coupled with mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is reported as an alternative and potentially useful method for the simultaneous analysis of various classes of phytohormones with diversified structures, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid
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Capillary electrophoresis (CE) coupled with mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is reported as an alternative and potentially useful method for the simultaneous analysis of various classes of phytohormones with diversified structures, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA), zeatin (Z), N6-benzyladenine (BA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The key to the CE-MS/MS analysis was based on electroosmotic flow reversal using a cationic polymer-coated capillary. Under optimum conditions, a baseline separation of eight phytohormones was accomplished within 30 min using 60 mM ammonium formate/formic acid buffer of pH 3.8 with −20 kV as the separation voltage. The accessibility of MS/MS together with the characterization by migration properties obtained by CE allows for the development of CE-MS/MS as an emerging potential method for the analysis of different classes of phytohormones in a single run. The utility of the CE-MS/MS method was demonstrated by the comprehensive screening of phytohormones in coconut (Cocos nucifera L.) water after pre-concentration and purification through solid-phase extraction (SPE) cartridge. IAA, ABA, GA and Z were detected and quantified in the purified coconut water extract sample. Full article
(This article belongs to the Special Issue Advances in Hyphenated Methods in Separation)

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Open AccessTechnical Note High-Throughput Mass Spectrometry Applied to Structural Genomics
Chromatography 2014, 1(4), 159-175; doi:10.3390/chromatography1040159
Received: 1 July 2014 / Revised: 3 September 2014 / Accepted: 11 September 2014 / Published: 9 October 2014
PDF Full-text (1100 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Mass spectrometry (MS) remains under-utilized for the analysis of expressed proteins because it is inaccessible to the non-specialist, and sample-turnaround from service labs is slow. Here, we describe 3.5 min Liquid-Chromatography (LC)-MS and 16 min LC-MSMS methods which are tailored to validation and
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Mass spectrometry (MS) remains under-utilized for the analysis of expressed proteins because it is inaccessible to the non-specialist, and sample-turnaround from service labs is slow. Here, we describe 3.5 min Liquid-Chromatography (LC)-MS and 16 min LC-MSMS methods which are tailored to validation and characterization of recombinant proteins in a high throughput structural biology pipeline. We illustrate the type and scope of MS data typically obtained from a 96-well expression and purification test for both soluble and integral membrane proteins (IMPs), and describe their utility in the selection of constructs for scale-up structural work, leading to cost and efficiency savings. We propose that value of MS data lies in how quickly it becomes available and that this can fundamentally change the way in which it is used. Full article
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