Two-dimensional capillary liquid chromatography: pH Gradient ion exchange and reversed phase chromatography for rapid separation of proteins

Pepaj, Milaim; Wilson, Steven Ray; Novotná, Kateřina; Lundanes, Elsa; Greibrokk, Tyge

doi:10.1016/j.chroma.2006.02.031

Cited by 71 publications

(62 citation statements)

References 29 publications

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“…In contrast, RPLC has advantages of high efficiency and high resolution, and more importantly, the mobile phase is compatible with MS, demonstrating preferable attributes for peptide separation [21][22][23]. Therefore, considering the merits of each mode and the inherent benefits of IMER, herein we developed an integrated platform by coupling CIEF-IMER-nanoreversedphase liquid chromatography (nanoRPLC) with MS for online protein profiling.…”

Section: Tingting Wangmentioning

confidence: 97%

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Wang

et al. 2011

Electrophoresis

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An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29 μg, was analyzed, and sequence coverages for BSA, myoglobin, β-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.

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Section: Tingting Wangmentioning

confidence: 97%

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Wang

et al. 2011

Electrophoresis

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“…[15][16][17][18] In addition, multiple combinations of the characterization techniques have been developed, e.g. LC-PDA-MS, 19 LC-NMR-MS, 20,21 LC-SPE-NMR, 22 LC-PDA-IR-NMR-MS, 23 and 2D-LC(IEC-RP)-MS. 24 These improve the sample separation, structural characterization and elucidation, and also the detection of valuable minor components in natural sources.…”

Section: Introductionmentioning

confidence: 99%

Automated molecular formula determination by tandem mass spectrometry (MS/MS)

Jarussophon

Acoca

Gao

et al. 2009

Analyst

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Automated software was developed to analyze the molecular formula of organic molecules and peptides based on high-resolution MS/MS spectroscopic data. The software was validated with 96 compounds including a few small peptides in the mass range of 138-1569 Da containing the elements carbon, hydrogen, nitrogen and oxygen. A Micromass Waters Q-TOF Ultima Global mass spectrometer was used to measure the molecular masses of precursor and fragment ions. Our software assigned correct molecular formulas for 91 compounds, incorrect molecular formulas for 3 compounds, and no molecular formula for 2 compounds. The obtained 95% success rate indicates high reliability of the software. The mass accuracy of the precursor ion and the fragment ions, which is critical for the success of the analysis, was high, i.e. the accuracy and the precision of 850 data were 0.0012 Da and 0.0016 Da, respectively. For the precursor and fragment ions below 500 Da, 60% and 90% of the data showed accuracy within #0.001 Da and #0.002 Da, respectively. The precursor and fragment ions above 500 Da showed slightly lower accuracy, i.e. 40% and 70% of them showed accuracy within #0.001 Da and #0.002 Da, respectively. The molecular formulas of the precursor and the fragments were further used to analyze possible mass spectrometric fragmentation pathways, which would be a powerful tool in structural analysis and identification of small molecules. The method is valuable in the rapid screening and identification of small molecules such as the dereplication of natural products, characterization of drug metabolites, and identification of small peptide fragments in proteomics. The analysis was also extended to compounds that contain a chlorine or bromine atom.

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“…These methods may use an approach based upon fractionation of intact proteins [6,9] or alternatively fractionation of the protein content of a cell following total digestion of the proteins into peptides [5,10,11,12]. Both of the approaches have been used in previous work for analysis of small amounts of sample.…”

Section: Introductionmentioning

confidence: 99%

“…A number of methods have been developed to fractionate the protein content of a cell on a micro-proteomic scale [5,6,7,8]. These methods may use an approach based upon fractionation of intact proteins [6,9] or alternatively fractionation of the protein content of a cell following total digestion of the proteins into peptides [5,10,11,12].…”

Section: Introductionmentioning

confidence: 99%

Micro-proteome analysis using micro-chromatofocusing in intact protein separations

Kim

Lubman

2008

Journal of Chromatography A

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Multidimensional liquid-based separation is required for fractionation and mapping of complex protein mixtures from cells. A method that has been used as the first dimension in such separations is chromatofocusing, which is a based on generating a pH gradient on an anion exchange column. The use of pH in the first dimension is essential where pH is a fundamental property of proteins and can provide information on post-translationally modified forms of a protein. In this work, a microchromatofocusing technique is introduced which can separate microgram levels of proteins from cell lysates for further analysis by LC-MS/MS. It is shown that this method can analyze 10 µg of sample and detect nearly 700-800 proteins from ovarian cancer cell line lysates.

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Two-dimensional capillary liquid chromatography: pH Gradient ion exchange and reversed phase chromatography for rapid separation of proteins

Cited by 71 publications

References 29 publications

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Automated molecular formula determination by tandem mass spectrometry (MS/MS)

Micro-proteome analysis using micro-chromatofocusing in intact protein separations

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