A novel online three-dimensional RP-SCX-RP platform for PC12 protein profiling

This journal supplement is the 60th ASMS Conference ProgramPoster presentation - Proteomics: Sample Preparation and Separations: TP 147The 60th ASMS Conference on Mass Spectrometry and Allied Topics, Vancouver, Canada, 20-24 May 2012. In Journal of The American Society for Mass Spectrometry, 2012, v. 23 suppl. 1, p. S92, abstract no. TP 14

Development of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach

Previously, we described an online high-/low-pH RP-RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP-RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP-RP separation. Relative to the RP-RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample. © 2011 Elsevier B.V.link_to_subscribed_fulltex

A fully automatable two-dimensional HILIC-RP liquid chromatography system with online tandem mass spectrometry for shotgun proteomics

Poster presentation - Proteomics: Sample Preparation and Separations: TP 148This journal supplement is the 60th ASMS Conference ProgramThe 60th ASMS Conference on Mass Spectrometry and Allied Topics, Vancouver, Canada, 20-24 May 2012. In Journal of The American Society for Mass Spectrometry, 2012, v. 23 suppl. 1, p. S92, abstract no. TP 14

Simultaneous analysis of polar and non-polar pesticides in one run using orthogonal LC separation

This journal supplement is the 59th ASMS Conference ProgramPoster Presentation - Environmental Analysis: Pharaceuticals and Pesticides: WP 325The 59th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO., 5-9 June 2011. In Journal of The American Society for Mass Spectrometry, 2011, v. 22 suppl. 1, p. 137, abstract no. WP 32

Fully automatable three-dimensional capillary-/nano-flow liquid chromatography for shotgun proteomics: a reversed-phase/strong cation exchange/reversed-phase approach

Poster presentation - Proteomics: Sample Preparation and Separations: TP 146This journal supplement is the 60th ASMS Conference ProgramThe 60th ASMS Conference on Mass Spectrometry and Allied Topics, Vancouver, Canada, 20-24 May 2012. In Journal of The American Society for Mass Spectrometry, 2012, v. 23 suppl. 1, p. S92, abstract no. TP 14

Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.link_to_subscribed_fulltex

Mechanistic examinations on isomerizations and dissociations of phosphorylated glycylserinyltryptophan (GpSW) radical cations

Poster presentation - Peptides: Framentation Mechanisms: TP 762This journal supplement is the 60th ASMS Conference ProgramThe 60th ASMS Conference on Mass Spectrometry and Allied Topics, Vancouver, Canada, 20-24 May 2012. In Journal of The American Society for Mass Spectrometry, 2012, v. 23 suppl. 1, p. S119, abstract no. TP 76

Roles of radicals and charges in the neutral H3PO4 loss of molecular phosphorylated peptide radical cations

Poster Presentation - Peprides: Fragmentation Mechanisms: WP 485This journal supplement is the 59th ASMS Conference ProgramThe 59th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO., 5-9 June 2011. In Journal of The American Society for Mass Spectrometry, 2011, v. 22 suppl. 1, p. 145, abstract no. WP 48

Fully automatable two-dimensional hydrophilic interaction liquid chromatography-reversed phase liquid chromatography with online tandem mass spectrometry for shotgun proteomics

We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) - using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) - with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC-reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC-reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 10 6 copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 10 4. This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.link_to_subscribed_fulltex