Relative Quantitation

Quantitative proteomics

To understand the functions of individual proteins, their relationships to each other and to complex biological systems, protein abundance changes relative to those of the relevant system must be measured. Discovery-based relative quantification using mass spectrometry (MS) is an analytical approach that allows researchers to determine relative protein abundance changes simultaneously and across multiple sample sets without having to know the identities of the proteins involved. Listed below are three commonly used techniques for relative quantitation of unknown protein and peptide targets via MS.

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During stable isotope labeling with amino acids in cell culture, or SILAC, biological samples are labeled in vitro with a heavy isotope version of the target amino acid. During protein synthesis, the natural light form of the amino acid is eventually replaced with its heavier version. Cells undergoing different experimental conditions (and in heavy or light media) can be mixed and all processing steps performed on the combined sample. This strategy reduces sample handling variability, resulting in more accurate quantitation. CID, ETD, EThcD, or HCD can be used for fragmentation.

Thermo Scientific tandem mass tag (TMT) reagents are isobaric chemical tags that offer an alternative to SILAC. Up to 10 targets can be tested simultaneously to identify and quantitate relative abundance changes in biological samples. In a typical workflow, different experimental conditions are labeled with different isobaric tags. The samples are then mixed and analyzed by LC-MS. When a labeled peptide is fragmented, each tag produces a unique reporter ion that is detected in the MS/MS spectrum. Protein quantitation is performed by comparing reporter ion intensities to controls.

This cost-effective alternative to SILAC and TMT compares protein abundance changes across multiple samples without isotopic labels. Samples are tested individually, and signals corresponding to unique peptide ions are integrated over the LC time scale, after which different runs are compared and common chromatographic features are aligned. Key benefits of label-free quantitation include the ability to compare unlimited numbers of samples, test samples of any origin, and identify peptides using different dissociations (CID, HCD, ETD, and EThcD).

Featured video

In this informative presentation, viewers gain a more complete picture of quantitiative proteomics using isobaric tags (known as TMT Multiplexing). A summary of steps involved, from sample preparation through instrument configuration to data analysis is discussed in depth to ensure analyst success.