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Global protein quantification

Quantitative proteomics includes powerful global discovery or targeted methods to analyze and understand protein dynamic changes in cells or tissues. Technological advances in instrumentation have increased the number of proteins that can be covered in a single sample up to several thousands.

Discovery proteomics can provide critical insights for our understanding of the global protein expression as well as modification profiles (e.g. phosphorylation) underlying the molecular mechanisms of biological processes and disease states. These quantitation methods include chemically labeling with isobaric labels using Tandem Mass Tags (TMT™, Thermo-Fisher) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ®, Sciex) suitable for all biological samples. Metabolic labeling using Stable Isotope Labeling with Amino Acids in Cell culture (SILAC) is suitable for labeling of proteins in cell cultures.

Quantitative mass spectrometry studies often follow a sequential workflow where methods such as fractionation by high-pH liquid chromatography (LC) are performed to reduce sample complexity prior to nanoLC. For plasma project we offer affinity depletion of high abundant proteins in human, rat and mouse plasma. (links to plasma depletion articles) Reduced complexity is a critical factor of peptide quantitation, as identification and quantification rates are directly proportional to sample complexity.
Data analysis for relative quantification is performed using Proteome Discoverer or MaxQuant.

TMT or iTRAQ

Relative quantitation with isobaric labeling (TMT and iTRAQ) is used to compare abundance of peptides and their corresponding proteins in multiple samples at MS/MS level. This methodology can also be applied to study post-translational modifications such as phosphorylation between multiple samples.

The proteins in up to ten samples are digested with trypsin and the peptides are labeled with different versions of the isobaric tags and then combined into one sample. Identical peptides, derived from different samples, are indistinguishable in their intact form during analysis. However, upon fragmentation in the MS instrument, each peptide variant produces a unique reporter ion used for quantification. Only peptides unique for a specific protein are considered for quantification. This multiplexed quantification reduces the variation between samples in the experiment and allow fold-change as low as ± 10-20% to be considered as significant results.

SILAC

Stable isotope labeling by amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of 'light' or 'heavy' forms of amino acids into proteins for quantitative proteomics.

In a SILAC experiment, differential labeling of two to three cell types can be performed where cells are grown with the natural amino acids, with 2H4-lysine and 13C6-arginine and with 15N213C6-lysine and 15N413C6-arginine. The samples are mixed, digested with trypsin and analyzed by nanoLC MS/MS. Since trypsin cleaves at the amino acids arginine and lysine all peptides are used for quantification. The labeling does not affect the chemical properties of the peptides and they co-elute from the nanoLC column but they remain distinguishable by MS. The peptide peaks of the differentially labeled samples can be very accurately quantified relative to each other to determine the peptide and corresponding protein ratios. This methodology can also be applied to study post-translational modifications as phosphorylation between multiple samples.

Researchers are required to perform the SILAC incorporation themselves. We offer consultation for setting the experimental design. Prior to large-scale experiments, incorporation testing by nanoLC MS analysis must be performed to determine the incorporation efficiency.

Page Manager: Jörgen Bergström|Last update: 3/29/2016
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