Current trends in differential expression proteomics: isotopically coded tags

doi:10.1016/S0167-7799(01)01793-0

Trends in Biotechnology

Volume 19, Issue 10, 1 October 2001, Pages S10-S16

https://doi.org/10.1016/S0167-7799(01)01793-0 Get rights and content

Abstract

Isotopically coded tag methodology holds significant promise for differential expression proteomic experiments. This methodology has the potential for high sensitivity, high coverage, and high throughput. Although significant technical advances have been made in the past year, this approach must be viewed as an emerging technique. Advances in sample fractionation, both at the protein and peptide level, and improved data acquisition schemes, will all be required before the full potential of the method is realized.

Section snippets

Differential expression proteomics and protein separations

Since Anderson's visionary proposal for the creation of the Human Protein Index in 1982 (Ref. 1), differential protein expression has become almost synonymous with 2D gel electrophoresis. As a method for the separation of intact proteins, 2D gels have no rivals, offering unparalleled separation efficiencies. The basis for this extremely high separation efficiency is its comprehensive multidimensional coupling of two different separation systems (isoelectric focusing and polyacrylamide gel

Isotope coded affinity tags

In 1999, Gygi et al. reported on an exciting new technology for differential expression proteomics – isotope coded affinity tags (ICAT) ⁵. This approach, from the laboratory of Ruedi Aebersold, uses a protein tag with three functional moieties – a cysteine reactive moiety, a linker with either eight hydrogens or eight deuteriums (giving an isotope code or mass tag of 8 Da), and a biotin moiety (the affinity tag). In this experiment (Fig. 1), the cysteine side chains in complex mixtures of

Challenges and opportunities with ICAT

Although the initial report demonstrated the utility of the ICAT approach, several challenges were also apparent. The first of these challenges relates to the cysteine labeling. Because the technique is based on labeling the cysteine residue(s) of a protein, proteins that do not contain a cysteine will not be detected with ICAT. In the yeast proteome, 8% of the proteins do not contain a cysteine. In other species, the percentage of proteins lacking a cysteine can be as high as 20% of the total

Sample fractionation schemes

Sample fractionation for ICAT will be best accomplished at both the protein level and the peptide level. Fractionation at the peptide level is the most straightforward approach for two reasons. First, there is a variety of well-established techniques for peptide fractionation for use with MS analysis. Second, as labeling of the peptides with the ICAT reagent will occur before this fractionation, the effects of losses from the fractionation step are minimized because the relative expression

Data acquisition schemes

Further enhancement of ICAT analysis will be made through the use of improved data acquisition schemes. All work published to date collects MS–MS data from peptides regardless of their differential expression levels. Assuming less than 10% of the proteins in any experiment are actually differentially expressed, such a data acquisition scheme spends 90% of the time acquiring data from non-biologically relevant proteins (non-differentially expressed proteins). The concept of differentially

Matrix-assisted laser desorption ionization

To date, most ICAT experiments have used electrospray ionization interfaces (ESI) online with nanoscale capillary LC systems. An alternate interface that presents significant advantages to ICAT experiments is matrix-assisted laser desorption ionization (MALDI). Both ESI and MALDI are commonly used with a variety of tandem mass spectrometers for qualitative proteomic experiments. The initial report on the use of a MALDI–Q-TOF (quadrupole time-of-flight) mass spectrometer for ICAT experiments was

Concluding remarks

ICAT must be viewed as an emerging technique for differential expression proteomics, and its true utility has yet to be fully evaluated. Advances in sample fractionation at the protein level, sample fractionation at the peptide level, and improved data acquisition schemes, will all be required for the full potential of ICAT to be realized. ICAT offers several advantages over 2D gels, but it also has many disadvantages. ICAT is amenable for the analysis of several classes of proteins that are

References (27)

J. Ji
Strategy for qualitative and quantitative analysis in proteomics based on signature peptides
J. Chromatogr. B Biomed. Sci. Appl.
(2000)
G.J. Opiteck
Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping
Anal. Biochem.
(1998)
S.P. Gygi
Measuring gene expression by quantitative proteome analysis
Curr. Opin. Biotechnol.
(2000)
M.T. Davis
Automated LC–LC-MS–MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses
J. Chromatogr. B Biomed. Sci. Appl.
(2001)
N.G. Anderson et al.
The Human Protein Index
Clin. Chem.
(1982)
J. Klose et al.
Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome
Electrophoresis
(1995)
J. Link
Direct analysis of protein complexes using mass spectrometry
Nat. Biotechnol.
(1999)
M.P. Washburn
Large-scale analysis of the yeast proteome by multidimensional protein identification technology
Nat. Biotechnol.
(2001)
S.P. Gygi
Quantitative analysis of complex protein mixtures using isotope-coded affinity tags
Nat. Biotech.
(1999)
X. Yao
Proteolytic ¹⁸O labeling for comparative proteomics: model studies with two serotypes of adenovirus
Anal. Chem.
(2001)

J.C. Giddings

Concepts and comparisons in multidimensional separation

J. High Resolut. Chromatogr. Chromatogr. Commun.

(1987)

G.J. Opiteck

Two-dimensional microcolumn HPLC coupled to a single-quadrupole mass spectrometer for the elucidation of sequence tags and peptide mapping

J. Microcolumn Sep.

(1998)

D.R. Goodlett

Strategies and methods for proteome analysis

Cited by (78)

The Use of Proteomics in the Study of Molecular Responses and Toxicity Pathways in Biological Systems
2011, Advances in Molecular Toxicology
Citation Excerpt :
Indeed, many proteomic studies have not been targeted to probe possible alterations of any particular function or molecular process in the biological system of interest. Thus, comparisons of protein profiles between samples from control and toxin-exposed living systems often represent a general feature of proteomic studies, an experimental setting that is termed “differential expression proteomics” [9,15,16]. By this term, it is then intended that analyses of proteomes are aimed at identification of qualitative and quantitative changes in the levels of proteins associated with distinct conditions of a biological system, independently of the fact that they might result from normal and/or pathological functioning, or else as a consequence of exogenous or endogenous perturbations of the system.
The characterization of the chains of events triggered by noxious agents in sensitive systems is mandatory for a full understanding of their mechanisms of toxicity and the advancement of mechanistic-based toxicity testing. The complexity inherent into the characterization of toxicity pathways and the identification of biomarkers representing nodes of altered cell functioning support the development of analyses at a system level. Proteomics is being increasingly used for the characterization of biological systems from a toxicological perspective. Differential expression proteomics has been employed in the area of microalgal toxins, and critical review of data indicates that system level analysis by this nonbiased approach allows identification of components of toxicity pathways. Still, the characterization of elements of the system has often involved targeted investigations, supporting the conclusion that the integration of systemic and reductionistic approaches will be essential to obtain quantitative, robust models of toxicity pathways and toxicity networks.
A new protocol of analyzing isotope-coded affinity tag data from high-resolution LC-MS spectrometry
2007, Computational Biology and Chemistry
Isotope-coded affinity tags (ICAT) is a labeling technique that provides insights into quantitative molecular changes. In this paper, we propose a new protocol to identify and analyze ICAT labeled peak pairs in high-resolution LC–MS data. Our major contributions are: (1) we use isotope distance constraint, ICAT distance constraint, and LC-span constraint to identify ICAT labeled peak pairs and (2) we propose to trigger tandem MS/MS scanning based on the ratio estimation value of identified ICAT peak pairs instead of the peak intensity values. Compared with current approaches that choose peaks with high intensity values for tandem MS/MS scanning, the new protocol can improve the scanning efficiency and accuracy.
Leveraging protein purification strategies in proteomics
2007, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
The proteomic studies, although, tend to be analytical in nature, yet many strategies of preparative protein purification can be usefully employed in such studies. This review points out the importance of purification techniques which are capable of dealing with samples which are suspensions rather than clear solution, e.g. aqueous two phase partitioning, three phase partitioning, expanded bed chromatography, etc. The review also outlines the potential of non-chromatographic techniques in dealing with fractionation of proteomes. Separation protocols which can deal with post-translationally modified (PTM) proteins are also considered.
Functional Genomics of Wine Yeast Saccharomyces cerevisiae
2007, Advances in Food and Nutrition Research
Citation Excerpt :
These technologies are not as robust as transcript profiling since proteins are far more heterogeneous than RNA. Currently, it is not possible to know with any degree of certainty that all proteins are being visualized and that relative ratios of proteins in the cytoplasm are being maintained through sample preparation and protein separation (Gygi et al., 1999a,b, 2000; Moseley, 2001; Peng et al., 2003; Ravichandran and Sriram, 2005). However, as with proteomics, some important observations have been made using subsets of components that could be accurately quantified.
The application of genomic technologies to the analysis of wine strains of Saccharomyces cerevisiae has greatly enhanced our understanding of both native and laboratory strains of this important model eukaryote. Not only are differences in transcript, protein, and metabolite profiles being uncovered, but the heritable basis of these differences is also being elucidated. Although some challenges remain in the application of functional genomic technologies to commercial and native strains of S. cerevisiae, recent improvements, particularly in data analysis, have greatly extended the utility of these tools. Comparative analysis of laboratory and wine isolates is refining our understanding of the mechanisms of genome evolution. Genomic analysis of Saccharomyces in native environments is providing evidence of gene function to previously uncharacterized open reading frames and delineating the physiological parameters of ecological niche specialization and stress adaptation. The wealth of information being generated will soon be utilized to construct commercial stains with more desirable phenotypes, traits that will be designed to be genetically stable under commercial production conditions.
Blood-brain barrier genomics and proteomics: elucidating phenotype, identifying disease targets and enabling brain drug delivery
2006, Drug Discovery Today
The blood–brain barrier (BBB) regulates the passage of material between the bloodstream and the brain. Recent genomic and proteomic studies of the BBB have identified some of the unique molecular characteristics of this vascular bed, and have reinforced the concept that the BBB is heavily involved in brain function. Genomic and proteomic techniques have also been used to analyze the molecular events underlying diseases that have BBB involvement, such as multiple sclerosis, Alzheimer's disease, stroke and HIV-1 infection. It is expected that a better understanding of the complex mechanisms that link the BBB to neurological disease will ultimately lead to the development of innovative treatments.
Endogenous transforming growth factor-β receptor-mediated Smad signaling complexes analyzed by mass spectrometry
2006, Molecular and Cellular Proteomics
Citation Excerpt :
In LC-MALDI MS TOF/TOF, more ribosome proteins were identified. The differences in identified protein numbers and types represent the differences in which the three analytical methods take advantage of the different properties of the peptides and their product ions (40). To verify interactions of Smad2 protein with its partners, co-immunoprecipitation and Western blot were used.
ASmad proteins are the central feature of the transforming growth factor-β (TGF-β) intracellular signaling cascade. They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes. Changes in Smad proteins and their complexes upon treatment with TGF-β were studied in mink lung epithelial (Mv1Lu) cell cultures. A time course of incubation with TGF-β was carried out to determine the peak of appearance of phosphorylated Smad2. Immobilized monoclonal antibody against Smad2 was then used to isolate the naturally occurring complexes. Three strategies were used to identify changes in proteins partnering with Smad2: separation by one-dimensional SDS-PAGE followed by MALDI peptide mass fingerprinting, cleavable ICAT labeling of the protein mixtures analyzed by LC-MS/MS, and nano-LC followed by MALDI MS TOF/TOF. Smad2 forms complexes with many other polypeptides both in the presence and absence of TGF-β. Some of the classes of proteins identified include: transcription regulators, proteins of the cytoskeletal scaffold and other tethering proteins, motility proteins, proteins involved in transport between the cytoplasm and nucleus, and a group of membrane adaptor proteins. Although some of these have been reported in the literature, most have not been reported previously. This work expands the repertoire of proteins known to participate in the TGF-β signal transduction processes.

View all citing articles on Scopus

View full text

Trends in Biotechnology

ReviewCurrent trends in differential expression proteomics: isotopically coded tags

Abstract

Section snippets

Differential expression proteomics and protein separations

Isotope coded affinity tags

Challenges and opportunities with ICAT

Sample fractionation schemes

Data acquisition schemes

Matrix-assisted laser desorption ionization

Concluding remarks

J. Chromatogr. B Biomed. Sci. Appl.

Anal. Biochem.

Curr. Opin. Biotechnol.

J. Chromatogr. B Biomed. Sci. Appl.

The Human Protein Index

Clin. Chem.

Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome

Electrophoresis

Direct analysis of protein complexes using mass spectrometry

Nat. Biotechnol.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology

Nat. Biotechnol.

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Nat. Biotech.

Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus

Anal. Chem.

Concepts and comparisons in multidimensional separation

J. High Resolut. Chromatogr. Chromatogr. Commun.

Two-dimensional microcolumn HPLC coupled to a single-quadrupole mass spectrometer for the elucidation of sequence tags and peptide mapping

J. Microcolumn Sep.

Strategies and methods for proteome analysis

Review
Current trends in differential expression proteomics: isotopically coded tags

Proteolytic ¹⁸O labeling for comparative proteomics: model studies with two serotypes of adenovirus