Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

Abstract

Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in peptide fractionation amenable to MS analysis are as well discussed.

The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis

“…Previous attempts to characterize the PM proteomes of mycorrhizal and nonmycorrhizal M. truncatula roots have been hindered by the lack of protein abundance measurement and incomplete genome resources (Valot et al 2006). Although, because of their hydrophobicity and relatively low abundance, the inventory of membrane proteins has lagged behind the survey of soluble proteomes, membrane proteomics has benefitted in recent years from technical advances in mass spectrometry (MS), bioinformatics resources, and label-free quantification methods (Abdallah et al 2012(Abdallah et al , 2014.…”

Proteomic profiling reveals candidate markers for arsenic-induced skin keratosis

“…Arsenical hyperkeratosis appears predominantly on the palms of the hands and soles of the feet (Liu et al, 2002). Two-dimensional gel electrophoresis has been applied in arsenic-related proteomic studies (Yu et al, 1993), but this method has the disadvantages of low reproducibility, poor representation of low abundant proteins, inaccurate quantification, high labor and time requirements, and inability to analyze the membrane and hydrophobic proteins (Abdallah et al, 2012). Shotgun proteomics, which utilizes reverse phase separation of peptides instead of electrophoretic protein separation, has higher speed, sensitivity, accuracy, and throughput with lower sample consumption (Zhang et al, 2013).…”

Biochemical and proteomic investigation of bovine nasal secretion

“…This modern proteomic technology avoids the band broadening associated with many chromatographic steps, which can decrease resolution (Kislinger et al, 2005). The application of this procedure, however, also requires the manipulation of specimens with high purity to avoid contamination problems (Kislinger et al, 2005 andAbdallah et al, 2012).…”

“…It is made of a long and flexible hydrocarbon chain linked to an ionic polar head. The detergent molecules will bind through their hydrophobic hydrocarbon tail to hydrophobic amino acids, subsequently promoting denaturing by binding favours amino acid-detergent interactions over amino acid-amino acid interactions (Abdallah et al, 2012).…”

“…This method is able to resolve peptides using different fractionation strategies, which offer high-throughput analyses of the sample proteome and provide a snapshot of the major protein constituents (Abdallah et al, 2012). This technology is also able to identify low-abundance proteins as well as highly acidic and basic proteins (Washburn et al, 2001).…”