Comprehensive Overview of Labeled and Label-Free Quantitative Proteomics Methods

Labeled Quantitative Proteomics

1.SILAC

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a technique for labeling proteins with stable isotopes under cell culture conditions. SILAC experiments involve incorporating essential amino acids (e.g., lysine (Lys) and arginine (Arg)) labeled with light, medium, or heavy stable isotopes into the growth medium. As the cells grow, newly synthesized proteins incorporate these labeled amino acids, thus becoming labeled with stable isotopes. After cell lysis, proteins are mixed in equal amounts and subjected to enzymatic digestion, followed by mass spectrometry (MS) analysis. Quantification is achieved by comparing the peak areas of labeled and unlabeled peptides in the MS spectra, and peptide sequencing is performed using tandem mass spectrometry (MS/MS).

 

2. Dimethyl Labeling

Dimethyl labeling is another isotopic protein labeling method, similar to SILAC. This technique involves chemically labeling the N-terminus or ε-amino group of lysine residues in proteins or peptides with different dimethyl tags after cell lysis. The MS analysis methods for SILAC and dimethyl labeling are essentially similar, providing comparable accuracy and analytical breadth in quantitative proteomics. SILAC generally exhibits higher reproducibility because the mixing of differentially labeled proteins occurs before proteolysis, whereas dimethyl labeling involves post-digestion peptides, leading to slightly lower reproducibility due to variations in system stability. Nevertheless, dimethyl labeling is cost-effective and increasingly used.

 

3. ICAT

Isotope-Coded Affinity Tag (ICAT) technology uses ICAT reagents to separate proteins. The ICAT tags vary in mass due to different isotopic compositions and can label cysteine residues in proteins from different cellular states. The reagent consists of three parts: a linker segment with eight hydrogen or deuterium atoms (light or heavy reagent), a thiol-reactive group specific to cysteine, and a biotin tag for affinity purification. The labeled proteins are digested, and the peptide complexes are purified via affinity chromatography. Only the isotopically labeled peptides are retained and subjected to MS analysis, where the signal intensities of light and heavy labeled peptides indicate relative protein expression levels.

 

The advantage of ICAT lies in its ability to directly analyze mixed samples (e.g., from normal and diseased cells or tissues) and rapidly identify low-abundance proteins, including membrane proteins. It can quantify the entire cellular proteome and specific subcellular components, such as mitochondria.

 

4. ¹⁸O Labeling

Enzyme-catalyzed ¹⁸O isotopic labeling involves incorporating ¹⁸O into peptides during enzymatic digestion, typically using trypsin. This method introduces oxygen atoms into the peptide backbone, achieving isotopic labeling by incorporating ¹⁸O from water during proteolysis.

 

5. iTRAQ

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) label the N-terminus and lysine residues of post-digestion peptides. iTRAQ reagents consist of a reporter group, a balance group, and a peptide reactive group. The reporter group has different mass numbers, and the balance group ensures the total mass remains constant, maintaining consistent retention times in chromatography. iTRAQ allows simultaneous labeling and quantitation of up to eight samples, providing relative quantitation based on the reporter ion intensities in the MS/MS spectra.

 

6. TMT

Tandem Mass Tags (TMT) technology uses isobaric tags to label peptides from different sources and quantify them via MS/MS. TMT reagents include a reporter group, a balance group, and a peptide reactive group, ensuring the same mass for different labels. In MS, labeled peptides exhibit the same mass-to-charge ratio in the MS1 spectrum, and fragmentation releases reporter ions with different mass numbers, enabling quantitation.

 

Label-Free Quantitative Proteomics

Label-free quantitation (LFQ) allows direct protein quantitation based on MS signal intensity without using labeling reagents. This method is versatile, suitable for various sample types, and widely used in quantitative proteomics. LFQ techniques include Extracted Ion Chromatogram (XIC) and Spectral Counting (SC).

 

1. XIC

XIC-based quantitation extracts the signal intensity of peptide ions over the retention time, quantifying using the XIC area or total intensity. Accurate XIC calculation can be achieved through AMT database matching or reverse protein database searching.

 

2. SC

Spectral counting quantifies proteins based on the number of identified peptide spectra, assuming higher protein abundance results in more detected peptides. This method is straightforward and widely applied in LFQ.

 

Proteomics involves the systematic analysis of proteins expressed by cells or tissues using techniques for separation, identification, quantification, and bioinformatics, with MS being a key tool. Quantitative proteomics, whether labeled or label-free, provides deep insights into the responses of cells and tissues to internal and external perturbations. The choice of an appropriate quantitation method depends on the sample type and experimental goals to ensure reliable and comprehensive proteomics analysis.