The dynamic range of protein expression in complex organisms coupled with the stochastic nature of discovery-driven tandem mass spectrometry (MS/MS) analysis continues to impede comprehensive sequence analysis and often provides only limited information for low-abundance proteins. High-performance fractionation of proteins or peptides prior to mass spectrometry analysis can mitigate these effects, though achieving an optimal combination of automation, reproducibility, separation peak capacity, and sample yield remains a significant challenge. Here we demonstrate an automated nanoflow 3-D liquid chromatography (LC)-MS/MS platform based on high-pH reversed phase (RP), strong anion exchange (SAX), and low-pH reversed phase (RP) separation stages for analysis of complex proteomes. We observed that RP-SAX-RP outperformed RP-RP for analysis of tryptic peptides derived from Escherichia coli and enabled identification of proteins present at a level of 50 copies per cell in Saccharomyces cerevisiae, corresponding to an estimated detection limit of 500 amol, from 40 μg of total lysate on a low-resolution 3-D ion trap mass spectrometer. A similar study performed on a LTQ-Orbitrap yielded over 4000 unique proteins from 5 μg of total yeast lysate analyzed in a single, 101 fraction RP-SAX-RP LC-MS/MS acquisition, providing an estimated detection limit of 65 amol for proteins expressed at 50 copies per cell.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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