Quantitative proteomics using mass spectrometry
Introduction
The complete sequences of more than 100 genomes and the nearly complete sequences of many others, including the human (http://www.nlm.nih.gov), are giving a new vision to the study of biological systems. However, function is usually fulfilled from the proteins containing the actual information rather than the potential as indicated by the genes. Furthermore, gene expression varies with time and is different in different cell types within an organism; as a result, the proteome is by far more complex than the genome. Often, gene expression has been studied using mRNA arrays. However, in view of recent studies showing that there is no good correlation between mRNA quantities and protein quantities 1., 2., 3., 4.• we ought to investigate biological problems also at the proteome level. It should be emphasized that post-translational modifications, regulation of protein function by proteolysis, and composition of macromolecular complexes can only be determined at the protein level.
Most of the initial efforts in proteomics were focused on methods to effectively identify large numbers of proteins. Recent developments in mass spectrometry (MS) enable high-throughput identification of proteins. However, to understand the function of a protein, changes in gene expression often have to be determined, thus quantitative protein profiling is an essential part of proteomics. For this purpose, several technologies to identify and quantify proteins from biological samples have been developed.
Two-dimensional gel electrophoresis (2DE) in combination with MS has been used for many years for studying the proteome (see [5] for a recent review). The development of a two-colour fluorescent labeling system, which allows two protein samples (e.g. control and disease) to be differentially labeled and analysed in the same gel, obviates many of the reproducibility problems inherent to 2DE (see [6] for a review). Even using this system, however, quantitative results might sometimes be ambiguous because of the presence of more than one protein in a single spot. Moreover, 2DE is labour intensive and difficult to automate.
Protein array technology has been recently developed and used for identifying novel ligands and for quantitative profiling (see [7] for a review). Although this approach has been used successfully, keeping proteins in their native and active conformations is still a challenge.
MS has played a major role in quantitative proteomics, and here we review the recent developments.
Section snippets
The role of stable isotopes for quantitation
In quantitative proteomics, the main sources of error are variations in the sample preparation procedures (e.g. protein extraction, enrichment, fractionation) and/or variations in the analysis (in this case MS). These errors can be significantly reduced using internal standards. An important characteristic of a standard is that its chemical–physical characteristics should be as similar as possible to the analyte to be analysed. For this reason, stable isotopes have been used since the early
Quantitation of post-translational modification level of proteins
Many biological processes are regulated by post-translational modifications. Phosphorylation and glycosylation are amongst the most common modifications and MS has been successfully used for their identification. In many cases, however, phosphorylation is a dynamic process with complex kinetics involving several amino acids within a single protein (for examples see http://www.lecb.ncifcrf.gov/phosphoDB/). Some of the in vitro pre-digestion and post-digestion labeling procedures described in
Conclusions
Current quantitative proteomics using MS is mainly based on the incorporation of stable isotope tags. Although numerous reports regarding protein quantitation using MS have been published, there are still many areas that require further developments (see Table 1 for a list of pros and cons).
In vivo labeling procedures are potentially the most accurate because the label is introduced early in the process; however, it is limited to cells that can be grown in culture. The advantage of the
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
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Cited by (94)
Improvements for absolute quantitation using electrochemical mass spectrometry
2019, International Journal of Mass SpectrometryCitation Excerpt :Therefore, accurate MS quantitation often has to use reference standards for calibration or isotope-labeled compounds as a reference [9,10]. To date, many quantitative MS methods have been developed with great successes, such as isotope-coded affinity tags (ICAT) [11–17], stable isotope labeling with amino acids in cell culture (SILAC) [15,18–21], isobaric tags for relative and absolute quantification (iTRAQ) [22–25], metal element chelated tags (MECT) [26] and isotope-coded protein labeling (ICPL) [27] etc. However, the reference standards or isotope-labeled compounds that are required for quantification might not be readily available, and sometimes their sytheses are cost prohibitive and time-consuming [28–31].
N-mustard analogs of S-adenosyl-l-methionine as biochemical probes of protein arginine methylation
2015, Bioorganic and Medicinal ChemistryCitation Excerpt :The desired m/z values for peptide modified by both 2 and 3 were observed (Supplemental Figs. S7 and S8, respectively), as well as the substantial increase in relative abundance for peptide modified with 4, compared to unmodified peptide (Fig. 3A). Quantification of peptide products was not conducted,24,25 as preparing analog-modified peptide standards were not synthetically feasible. Alternative quantification methods using deuterated analogs are being explored for future studies.16
Effect of apodization on FT-ICR mass spectrometry analysis of petroleum
2014, International Journal of Mass SpectrometryCitation Excerpt :The relationship between resolving power and peak skirt reduction for complex mixture must be optimized. Moreover, the effect of apodization on the peak intensity should be addressed if FT-ICR MS is to be used as a quantitative analytical method [31–34]. The concept of window functions should be re-evaluated to optimize apodization during complex mixture FT-ICR MS.
Proteomics
2012, Encyclopedia of Applied Ethics: Volume 1-4, Second EditionApplications in Data-Intensive Computing
2010, Advances in ComputersCitation Excerpt :A wide variety of technologies are being used in the field of proteomics. One of the most widely used technologies is known as “shotgun” or “bottom-up” proteomics, in which a complex protein mixture is digested with a protease and analyzed with tandem mass spectrometry (MS/MS) coupled with LC [75–80]. In this approach, digested pieces of the proteins, called peptides, are passed through a cylindrical liquid column.