Reference: Lesur A, et al. (2012) Peptides quantification by liquid chromatography with matrix-assisted laser desorption/ionization and selected reaction monitoring detection. J Proteome Res 11(10):4972-82

Reference Help

Abstract


We present a novel analytical platform for peptides quantitative assays in biological matrices based on microscale liquid chromatography fractionation and matrix-assisted laser desorption/ionization mass spectrometric detection using the selected reaction monitoring (SRM) mode. The MALDI source was equipped with a high frequency Nd:YAG laser (1000 Hz) and mounted on a triple quadrupole/linear ion trap mass spectrometer (MALDI-QqQ(LIT)). Compared to conventional LC-ESI-SRM/MS, the separated analytes are "time-frozen" onto the MALDI plate in fractions, and navigation through the LC chromatogram makes it possible to perform SRM experiments as well as enhanced product ion spectra acquisition for confirmatory analyses without time constraints. The LC spots were analyzed using different rastering speeds ranging from 0.25 to 4 mm/sec with the shortest analysis time of 425 ms/spot. Since the LC runs can be multiplexed and do not need a comprehensive investigation, the present platform offers a valuable alternative to LC-ESI-SRM/MS for high throughput proteomic analyses. In addition, the derivatization of the N-terminal α-amino group by sulfonation was found to be key for the fragmentation of singly charged peptides under low collision energy regime. Under such conditions, y-ion series were observed in the MS/MS spectra, and thus the design of SRM experiments was greatly simplified. The quantitative performance of the platform was compared to that of LC-ESI-SRM/MS by spiking yeast tryptic peptides in human plasma digests. Both platforms exhibited similar sensitivities, accuracy (within ±20%) and precision (under 20%) in the relative quantification mode. As a proof of principle, the relative and absolute quantification of proteins associated with glycolysis, glyoxylate and tricarboxylic acid (TCA) cycles over a growth time course of Saccharomyces cerevisiae on glucose media was successfully performed using isotopic dilution.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Lesur A, Varesio E, Domon B, Hopfgartner G
Primary Lit For
Additional Lit For
Review For

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

Disease 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 Disease Ontology Term Qualifier Evidence Method Source Assigned On 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 Direction Regulation Of Happens During Method Evidence

Post-translational Modifications


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 its 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.

Site Modification Modifier Reference

Interaction Annotations


Genetic Interactions

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 Allele Assay Annotation Action Phenotype SGA score P-value Source Reference

Physical Interactions

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 Assay Annotation Action Modification Source Reference

Functional Complementation 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 its 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 Species Gene ID Strain background Direction Details Source Reference