Reference: Zhou F, et al. (2011) Online nanoflow reversed phase-strong anion exchange-reversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms. Anal Chem 83(18):6996-7005

Reference Help

Abstract


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 ?g 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 ?g 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.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't | Research Support, N.I.H., Extramural
Authors
Zhou F, Sikorski TW, Ficarro SB, Webber JT, Marto JA
Primary Lit For
Additional Lit For
Review For

Interaction Annotations


Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.

Interactor Interactor Type Assay Annotation Action Modification Phenotype Source Reference

Gene Ontology Annotations


Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.

Gene Gene Ontology Term Qualifier Aspect Method Evidence Source Assigned On Annotation Extension Reference

Phenotype Annotations


Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details.

Gene Phenotype Experiment Type Mutant Information Strain Background Chemical Details Reference

Regulation Annotations


Increase the total number of rows displayed on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; to filter the table by a specific experiment type, type a keyword into the Filter box (for example, “microarray”); download this table as a .txt file using the Download button or click Analyze to further view and analyze the list of target genes using GO Term Finder, GO Slim Mapper, SPELL, or YeastMine.

Regulator Target Experiment Assay Construct Conditions Strain Background Reference