Quantitative Proteomics

General Technique

At the Institute for Systems Biology (ISB), quantitative proteomics technologies have been developed to comprehensively identify and quantify proteins in two or more complex samples. The techniques are based on the use of stable isotopes to differentially label proteins or peptides, and mass spectrometry to compare the relative abundance of the proteins in different samples. Once proteins are differentially labeled with stable isotopes, they can be distinguished during mass spectrometry by a characteristic mass shift. In addition, the relative abundance of isotopically-labeled peptide pairs can be determined by comparing the ion signal intensities of the peptides.

One approach to stable isotope tagging that we have developed takes advantage of Isotope Coded Affinity Tagging (ICAT) reagents. The ICAT reagents consists of a biotin affinity tag, a polyether linker that can incorporate stable isotopes (e.g., deuterium), and an iodoacetamide reactive group that specifically reacts with cysteinyl thiols. Stable isotopes are incorporated post isolation into proteins by selective alkylation of cysteines with either an isotopically heavy reagent that incorporates eight deuterium atoms in place of eight hydrogen atoms (i.e., d8-ICAT) or a light reagent containing the natural distribution of elements (i.e., d0-ICAT).

After labeling, protein samples are combined and digested with trypsin. Peptides are fractionated by strong cation exchange (SCX) chromatography and the biotinylated ICAT-labeled peptides are then purified by avidin chromatography. Peptides are further resolved by microcapillary reversed phase liquid chromatography , before mass spectrometry analysis using ion trap instruments, hybrid quadrupole time of flight (TOF) instruments, or TOF-TOF instruments. Because isotopically-labeled peptide pairs are virtually chemically identical, they co-elute during column chromatography.

The mass spectrometer toggles between survey scans (MS), and sequencing scans (MS/MS). The ratio of ion intensities from co-eluting ICAT-labeled peptides is determined in the survey scan and defines the ratio between parent proteins in the starting samples. Subsequent MS/MS analysis generates a spectrum that can be used by the algorithm SEQUEST to search a protein sequence database to identify the protein. Finally, search results are analyzed by probability-based algorithms, PeptideProphet and ProteinProphet, to estimate the likelihood that each peptide and protein is correctly identified. Relative quantitation is performed by ISB-developed software - Xpress and/or ASAPRAtio - that automatically integrates under the relevant regions of parent/single-ion trace chromatograms. Therefore, in a single analysis, stable isotope tagging and MS permits one to identify the proteins in two samples and quantify their relative abundances.

Purpose/use/application of the technique:

1) Global protein profiling. Global protein profiling experiments are performed to obtain a comprehensive description of the relative levels of protein expression in two or more samples. For example, the technique can be used to compare the changes in protein expression that acc ompany the transition from a normal cell state to a cancerous state. These experiments are an important compliment to gene expression measurements because they allow the detection of post-transcriptional events. For example, protein expression levels can be regulated during translation and protein activity can be regulated by numerous modifications such as phosphorylation and glycosylation. In a typical protein profiling experiment, protein extracts from two or more samples are differentially labeled with stable isotopes and analyzed as described above. More than one thousand proteins can be identified and quantified in a single analysis. When stable isotope labeling is combined with a strategy that allows isolation of specifically modified proteins/peptides, such as the glycocapture technique, in-depth information about the levels of specifically modified proteins in two or more samples can be obtained.

2) Macromolecular complex and organelle analysis. We have pioneered a powerful approach for characterization of macromolecular complexes that is based on the use of stable isotope tagging of proteins or peptides and MS to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. By comparing the enrichment ratios of peptides/proteins derived from a specific complex purification and a control purification, bona fide complex components are reliably distinguished from non-specifically co-purifying proteins. Importantly, because one-step affinity purifications can be used for sample purification, protein losses due to multiple purification steps are avoided, and the potential to identify weakly associated factors is increased. This is an important point when attempting to analyze complexes which are difficult to purify in sufficient quantities for MS analysis. The approach also permits the detection of dynamic changes in the composition of complexes. For example, quantitative changes in the composition of complexes isolated from extracts of cells grown under different condition can be detected. The important features of this approach are the following:

  1. It is comprehensive. The high resolving power of liquid chromatography combined with MS (LC-MS) permits the characterization of hundreds of peptides/proteins in a single analysis. Mass spectrometric analysis also permits the identification of post translational modifications.
  2. It is sensitive. Mass spectrometers can routinely detect low femtomole quantities of peptides.
  3. It is quantitative. This feature greatly facilitates the analysis of protein complexes.

3) Absolute protein quantification. For selective profiling of proteins in mixtures, we have developed methods that make use of isotopically labeled synthetic reference peptides that uniquely identify a particular protein. Addition of measured amounts of a panel of such reference peptides to a complex mixture of peptides enables precise quantification of selected peptides. The platform consists of a peptide separation module for the generation of ordered peptide arrays from the combined peptide sample on the sample plate of a MALDI mass spectrometer, a high throughput MALDI TOF/TOF mass spectrometer and a suite of software tools for the selective analysis of the targeted peptides and the interpretation of the results. The method was applied to the analysis of the human blood serum proteome to demonstrate its feasibility as a high throughput screening technology.

For absolute quantification of specific proteins in complex mixtures, we have developed a new class of reagents termed VICAT (visible isotope-coded affinity tags). Like ICAT, VICAT reagents tag thiol groups of cysteines, introduce a biotin affinity handle, a cleavable linker for removing a portion of the tag, and an isotope tag for distinguishing sample and internal standard peptides. The reagent also includes a visible moiety for tracking target peptides in a gel-based separation technique such as isoelectric focusing. This feature allows for rapid enrichment of target and internal standard peptides from a complex mixture. After isoelectric focusing, the sample is analyzed by μLC-ESI-MS/MS operating in selected reaction monitoring mode, to determine the absolute abundance of a specific peptide in a cell lysate. The strategy was used successfully to determine the absolute abundance of human group V phospholipase A (a relatively low abundance protein) in a cell lysate. This approach should be useful for numerous applications including the analysis of candidate disease markers in complex mixtures such as serum.

Example(s) of projects at ISB that use this technique:

Ongoing area of technology development:

Yes, new isotopic labeling strategies are being developed and applied. One approach involves the use of a solid phase reagent that is capable of capturing and labeling cysteinyl peptides from complex mixtures. The solid phase reagent contains a sulfhydrl-reactive group connected via an isotopic tag to a glass bead-attached acid labile linker. The two forms of the reagent are distinguished by a leucine molecule containing either seven hydrogen atoms or seven deuterium atoms. Peptides derived from two samples are incubated with either form of the beads, and cysteinyl peptides are covalently captured. The beads are then combined, and washed to remove unbound peptides and other molecules. After exposure to strong acid the isotopically labeled peptides are released and recovered. Thus, in one operation, peptides are isotopically labeled, sample complexity is reduced and peptides are purified.

Because of these features, the solid phase method is simpler, more efficient and more sensitive than the ICAT method which involves two chromatography steps after labeling. It also is more economical. Another approach involves the use of amine-reactive iTRAQ reagents (Applied Biosystems, Inc.) to isotopically label peptides. iTRAQ reagents consist of three moieties:

The last two groups constitute an isobaric tag. There are four forms of the iTRAQ reagent, and each contains stable isotopes that are uniquely distributed between the reporter and balance groups such that all forms are isobaric. However, during MS/MS, the reporter group is released and generates an ion signal with a distinct m/z for each form of iTRAQ which is used to quantify the peptide to which it was attached. The reagent offers several advantages over previously used sulfhydryl-reactive ICAT reagents:

  1. Since amine groups are more abundant than cysteinyl sulfhydrl groups, iTRAQ enables quantitative analysis of more peptides/protein, thereby improving protein coverage and the accuracy of quantification. This feature also enables quantification of proteins that lack cysteines.
  2. Since the reagent comes in four forms it enables analysis of up to four distinctly labeled samples in a single MS experiment.
  3. The isobaric nature of the reagents may improve detection of peptides because the mass spectrum is simplified, and the ion signal for a peptide is a combination of the signals from each sample being analyzed.
  4. Quantification may be improved because it is based on detection of a reporter that fragments during MS/MS and therefore, does not rely on the reconstruction of ion chromatograms.
  5. The approach involves fewer chromatographic steps than the ICAT approach which will reduce sample losses and save time.

Representative publication(s):

Zhou, H., Ranish, J. A., Watts, J. D., and Aebersold, R., Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry, Nat Biotechnol, 20, 512 (2002).

Ranish, J. A., Yi, E. C., Leslie, D. M., Purvine, S. O., Goodlett, D. R., Eng, J., and Aebersold, R., The study of macromolecular complexes by quantitative proteomics, Nat Genet, 33, 349 (2003).

Himeda, C. L., Ranish, J. A., Angello, J. C., Maire, P., Aebersold, R., and Hauschka, S. D., Quantitative proteomic identification of six4 as the trex-binding factor in the muscle creatine kinase enhancer, Mol Cell Biol, 24, 2132 (2004).

Brand, M., Ranish, J. A., Kummer, N. T., Hamilton, J., Igarashi, K., Francastel, C., Chi, T. H., Crabtree, G. R., Aebersold, R., and Groudine, M., Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics, Nat Struct Mol Biol, 11, 73 (2004).

Ranish, J. A., Hahn, S., Lu, Y., Yi, E. C., Li, X. J., Eng, J., and Aebersold, R., Identification of TFB5, a new component of general transcription and DNA repair factor IIH, Nat Genet, 36, 707 (2004).

Baliga, N.S. et al. Systems Level Insights Into the Stress Response to UV Radiation in the Halophilic Archaeon Halobacterium NRC-1. Genome Res. 14, 1025-1035 (2004).

Baliga, N.S. et al. Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global systems approach. Proc Natl Acad Sci U S A 99, 14913-8. (2002). (Lin et al., 2005; Lu et al., 2004; Marelli et al., 2004; Pan et al., 2005; Tian et al., 2004)

Lin, B., White, J. T., Lu, W., Xie, T., Utleg, A. G., Yan, X., Yi, E. C., Shannon, P., Khrebtukova, I., Lange, P. H., Goodlett, D. R., Zhou, D., Vasicek, T. J., and Hood, L. (2005). Evidence for the presence of disease-perturbed networks in prostate cancer cells by genomic and proteomic analyses: a systems approach to disease. Cancer Res 65, 3081-91.

Lu, Y., Bottari, P., Turecek, F., Aebersold, R., and Gelb, M. H. (2004). Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags. Anal Chem 76, 4104-11.

Marelli, M., Smith, J. J., Jung, S., Yi, E., Nesvizhskii, A. I., Christmas, R. H., Saleem, R. A., Tam, Y. Y., Fagarasanu, A., Goodlett, D. R., Aebersold, R., Rachubinski, R. A., and Aitchison, J. D. (2004). Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane. J Cell Biol 167, 1099-112. Epub 2004 Dec 13.

Pan, S., Zhang, H., Rush, J., Eng, J., Zhang, N., Patterson, D., Comb, M. J., and Aebersold, R. (2005). High throughput proteome screening for biomarker detection. Mol Cell Proteomics 4, 182-90. Epub 2005 Jan 5.

Tian, Q., Stepaniants, S. B., Mao, M., Weng, L., Feetham, M. C., Doyle, M. J., Yi, E. C., Dai, H., Thorsson, V., Eng, J., Goodlett, D., Berger, J. P., Gunter, B., Linseley, P. S., Stoughton, R. B., Aebersold, R., Collins, S. J., Hanlon, W. A., and Hood, L. E. (2004). Integrated genomic and proteomic analyses of gene expression in Mammalian cells. Mol Cell Proteomics 3, 960-9. Epub 2004 Jul 6.


Alan Aderem




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