Dataset Information

Single shot CZE-MS/MS for phosphoproteomics

ABSTRACT: The dependence of capillary zone electrophoresis separations on the charge state of analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with advanced peak determination algorithm for phosphoproteomics analysis. A linear polyacrylamide coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume is between 100 nL and 150 nL of phosphopeptides dissolved in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single shot CZE-ESI-MS/MS based phosphoproteome analysis.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Zhenbin Zhang

LAB HEAD: Zhenbin Zhang

PROVIDER: PXD012888 | Pride | 2019-06-13

REPOSITORIES: Pride

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Publications

Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample.

Zhang Zhenbin Z Hebert Alexander S AS Westphall Michael S MS Coon Joshua J JJ Dovichi Norman J NJ

Journal of proteome research 20190626 8

The dependence of capillary zone electrophoresis (CZE) separations on the charge state of the analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume was between 100 and 150 nL of a ...[more]

PMID: 31180221

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Project description:A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes. 2 Biological replicate experimens conducted 1 month apart. In the first there were 2 dye-swapped duplicates (total 4) of VC+MeOH versus MeOH only. In the second experiment there was one set of dye swapped arrays. Thus 6 arrays were performed including biological replicates, dye swapped replicates and technical duplicates.

Dataset Information

Single shot CZE-MS/MS for phosphoproteomics

Publications

Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample.

Similar Datasets

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