2.2. Literature review of Saccharomyces cerevisiae proteomic analysis
2.2.7. The outlook of S. cerevisiae proteomics analysis
The analysis of the proteome is an important part of biological systems research since the expression of proteins can provide rich information for the understanding of biological processes. The study of protein abundances (relative and absolute), protein activities, subcellular location of proteins, proteins with modification, as well as the interaction of proteins in the network linkage maps to other proteins, or other types of biomolecules such as DNA, and lipids will predominate in next five years. This is because merely measuring the quantitative amounts of proteins alone is insufficient to explain the biology. Moreover, the application of different types of systematic measurements (such as the combination of mRNA expressions and protein expressions) in the same biological system, as well as more dynamic and temporal measurements will provide more useful information in studying complex biological processes.
Recently, the study of proteomic analysis of S. cerevisiae has received more attention, since this helps to reflect clearly the interaction between biochemical, cell biology and the specific functions of proteins. In addition, the development of MS/MS technology leads to the rapid identification of almost any protein in the S. cerevisiae genome for PTM study, and to investigate the S. cerevisiae interactome. This is important since the idea of proteome analysis is to identify and analyse a large number of proteins in a functional context. In addition, the high accuracy of the analysis of whole cell proteins is required to characterise and distinguish the difference of modified proteins forms which is difficult to obtain by peptide-level analysis. Moreover, most cellular processes in S. cerevisiae are regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues [120], and at least 30% of all proteins contain covalently bound phosphate [121].
Although this modification appears widely in S. cerevisiae proteomes, the identification of such sites in proteins is still challenging [121], even when carried out on highly purified proteins [122]. One reason for that is the negative charge of phosphopeptides, which reduces ion intensity, and since electrospray is generally operated in the positive mode, this means that these phosphopeptides are difficult to detect by mass spectrometry [123].
Another reason might be their hydrophilicity which can interfere with reverse-phase chromatography, and other factors [122, 124]. Therefore, the issues that are required to be overcome for generations of better MS equipment that seek application in the proteomics arena are those that can successfully analyse intact proteins (top-down), not with the currency of peptides (bottom-up) [80]. The bottom-up proteomics workflows do not allow for the study of combinations of site-specific mutations and PTMs in full, intact proteins, leading to the development of the top-down proteomics in which the exact molecular weight and direct fragmentationof the protein ions in the gas phase are enhanced [125].
The quantitative proteomic analysis of S. cerevisiae based on stable isotopic tags will predominate over that using 2-DE workflows in next few years, since these approaches have many advantages as detailed above. The in vitro labeling techniques such as ICAT, and especially iTRAQ will be applied widely in the study of the S. cerevisiae proteome. To date, both ICAT and iTRAQ have been successfully applied for proteome quantitation of S.
cerevisiae [11, 27-29, 68, 71]. With its advantages of 4 plex and soon 8 plex labeling, as well as the labeling of all peptides/proteins (not just those that contain cysteine), iTRAQ should be the preferable method for S. cerevisiae proteomic quantitation where a wider screen is necessary. Moreover, the absolute quantitation approach based on application of internal standards (for example synthetic peptides labeled with isotopic modified amino acids [13, 14]) has been developed and recently applied. This method offers an extremely accurate quantitative proteomic analysis of prior specified targets [125], allowing one to focus on only proteins functioning in more focussed metabolic targets in S. cerevisiae, such as glycolysis/gluconeogenesis and the TCA cycle. Absolute quantitation will become more
important generally in proteomics, taking over from the relative measures that are commonplace today.
Of course, the development of proteomics cannot continue without the improvement of MS/MS instrumentation. An improvement in speed, sensitivity, ease of operation, and purification procedures will be essential requirements for new generations of mass spectrometers [126]. For example, the combination of an extra trap column and a nano-LC- (RP)-MS/MS instrument will improve the detection of small peptides (low molecular weight, as well as short sequences) with low hydrophobicity. Moreover, since these proteins pass through the trap column easily when the injection of sample is performed, this leads to loss of these samples, therefore the addition of an extra trap column is necessary.
Another example is “intelligent” mass spectrometers, which can automatically move on to another ion if the current ions (peptides) have enough confidence for identification. This helps to reduce the time for scanning each ion (peptides), and increasing the chance for detection of other ion (peptides), as a result, more distinct ions (peptides, and then proteins) can be detected. Since the number of S. cerevisiae proteins is significant compared to many organisms, proteomic analysis is still incomplete. It means that current methods and techniques used for proteomic analysis are not powerful enough to identify and quantify simultaneously most of proteins covered by the genome in a single experiment. Therefore, the requirements of identification and quantitation of thousands of proteins, protein activities, and protein modifications will lead to the development of new methods such as reverse-phase columns for hydrophilic proteins, as well as increased MS intensity.
Moreover, new methods also must show significant benefits such as saving time, cost, reduction of preparation steps, as well as minimisation of number of experiments for a single experiment. As a prospect, the combination of shotgun proteomics and the new advanced mass spectrometry generations (such as the FT-ICR coupled with MS/MS) will provide powerful tools for detection, identification, and quantification of thousands of proteins from complex samples. These will offer a new horizon for S. cerevisiae proteomic
analysis. Moreover, quantitative proteomics approaches applied to S. cerevisiae (relative quantitation and especially absolute quantitation) will provide a powerful tool to complete (with high understanding of) the dynamics of protein-protein interaction maps of S.
cerevisiae, where measurement of these interactions have been only static so far. Once dynamic protein-protein interaction maps of S. cerevisiae are firmly established, finding the answers to questions on how proteins can be reorganised in response to changes of intracellular (or maybe intracellular) metabolites/(signals) will be simpler.
Moreover, to the authors’ knowledge there are likely to be a number of unknown components of S. cerevisiae that need to be discovered. This is in addition to putative proteins (approximately 1,000 genes (proteins) [109]) in the genome that have not been well characterised yet. Therefore, the characterization of these putative proteins will continue in the next few years.