Quantitative proteomics

Quantitative proteomics is mainly performed by two-dimensional gel electrophoresis (2-DE), preparative native PAGE, or mass spectrometry (MS).

However, a recent developed method of quantitative dot blot (QDB) analysis is able to measure both the absolute and relative quantity of an individual proteins in the sample in high throughput format, thus open a new direction for proteomic research.

[5] The concentration of a protein can be determined by measuring the OD at 280 nm on a spectrophotometer, which can be used with a standard curve assay to quantify the presence of tryptophan, tyrosine, and phenylalanine.

Classical 2-DE based on post-electrophoretic dye staining has limitations: at least three technical replicates are required to verify the reproducibility.

[citation needed] Mass spectrometry (MS) represents one of the main technologies for quantitative proteomics with advantages and disadvantages.

This technology has been used to label whole Saccharomyces cerevisiae cells,[12] and, in conjunction with mass spectrometry, helped lay the foundation of quantitative proteomics.

As with relative quantification using isotopic labels, peptides of equal chemistry co-elute and are analyzed by MS simultaneously.

A mathematically rigorous approach that integrates peptide intensities and peptide-measurement agreement into confidence intervals for protein ratios has emerged.

Metal-coded tags (MeCAT) method is based on chemical labeling, but rather than using stable isotopes, different lanthanide ions in macrocyclic complexes are used.

Thus it is possible to determine the absolute amount of protein down to attomole range using external calibration by metal standard solution.

Mass spectrometers have a limited capacity to detect low-abundance peptides in samples with a high dynamic range.

SILAC requires growing cells in specialized media supplemented with light or heavy forms of essential amino acids, lysine or arginine.

Because the peptides containing heavy and light amino acids are chemically identical, they co-elute during reverse-phase column fractionation and are detected simultaneously during MS analysis.

Recently, a new technique called NeuCode SILAC,[16] has augmented the level of multiplexing achievable with metabolic labeling (up to 4).

The increased multiplexing capability of NeuCode amino acids is from the use of mass defects from extra neutrons in the stable isotopes.

Protein or peptide samples prepared from cells, tissues or biological fluids are labeled in parallel with the isobaric mass tags and combined for analysis.

There are two different methods of quantification in label-free quantitative proteomics: AUC (area under the curve) and spectral counting.

This is limited, however, by the mass spectrometry software's ability to recognize and match peptide patterns of associations between the precursor and product ions.

It can be applied on a global proteome level, or on specifically isolating binding partners in pull-down or affinity purification experiments.

Thus, it has shown great promise in monitoring side-effects of small drug-like molecules and understanding the efficacy and therapeutic effect of one drug target over another.

Quantitative mass spectrometry.
Examples of quantitative proteomic workflows. Red represents physiological sample of interest, while blue represents control sample. White boxes represent areas where errors are most likely to occur, and purple boxes represent where the samples have been mixed. [ 8 ]
Work flow of the Quantification of the physiological differences in α and β cells in mice using computer prediction (A) and SILAC isotope-label quantification (B). (C) is the candidate list of kinases that indicate physiological differences in α and β cells. [ 21 ]