Liquid Chromatography–Mass Spectrometry-Based Quantitative Proteomics

Abstract

During the last decades, molecular sciences revolutionized biomedical research and gave rise to the biotechnology industry. During the next decades, the application of the quantitative sciences--informatics, physics, chemistry, and engineering--to biomedical research brings about the next revolution that will improve human healthcare and certainly create new technologies, since there is no doubt that small changes can have great effects. It is not a question of "yes" or "no," but of "how much," to make best use of the medical options we will have. In this context, the development of accurate analytical methods must be considered a cornerstone, since the understanding of biological processes will be impossible without information about the minute changes induced in cells by interactions of cell constituents with all sorts of endogenous and exogenous influences and disturbances. The first quantitative techniques, which were developed, allowed monitoring relative changes only, but they clearly showed the significance of the information obtained. The recent advent of techniques claiming to quantify proteins and peptides not only relative to each other, but also in an absolute fashion, promised another quantum leap, since knowing the absolute amount will allow comparing even unrelated species and the definition of parameters will permit to model biological systems much more accurate than before. To bring these promises to life, several approaches are under development at this point in time and this review is focused on those developments.

Functional proteomics to dissect tyrosine kinase signalling pathways in cancer

“…The ability of MS to resolve heavy and light isotopically labelled molecules has been extensively used to design approaches for quantitation [112][113][114][115] . The common labelling methods are: metabolic, typified by stable isotope labelling with amino acids in culture (SILAC) [116][117][118] .…”

Quantitation of glutathione S-transferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography–tandem mass spectrometry using isotope-labeled wing peptides as an internal standard

“…The candidate signature peptides should be specific to the target protein and avoid containing amino acids susceptible to chemical modifications, such as cysteine and methionine. Additionally, the selected signature peptides should have high MS signal intensity and can be reproducibly observed in digested samples [ 24 – 26 ]. In the present work, full scan LC–MS/MS analyses for the tryptic peptides from standard proteins (OsGSTF14 and OsGSTU6) and rice root samples using nanoLC-LTQ-Orbitrap were performed.…”

“…These disadvantages limit their large-scale application in protein quantification. In recent years, protein quantitative approach based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has become increasingly popular in target-proteomics studies, due to good accuracy, precision and high-throughput for protein quantification in high-complexity samples [ 24 – 26 ]. Multiple reaction monitoring mass spectrometry incorporated with stable isotope-labeled synthetic internal standard peptide is one of the LC–MS/MS-based protein quantitative approaches.…”

Divide and Conquer

“…These can be done with or without adding a mass tag to the peptide (tag- vs. label-free methods, respectively) 80 . Label-free methods base quantification on the signal intensity originated in the MS (peak area) or on the number of times a peptide is observed 81, 82 .…”