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Collecting and organizing systematic sets of protein data

Nature Reviews Molecular Cell Biology volume 7, pages 803–812 (2006)Cite this article

Key Points

  • Systems biology aims to develop experimentally-validated quantitative models of complex biochemical pathways. The data needed for this task cannot be generated by 'omics'-type methods alone, but instead require a hypothesis-driven approach that integrates multiple experimental techniques.

  • There are trade-offs to consider when choosing the types of assay to use in a systems biology study because throughput, multiplexing, sample size, sampling density and ease of use cannot all be simultaneously maximized. Experimental studies should be designed to be compatible with the chosen modelling approach.

  • For monitoring protein-signalling events, affinity-based methods, mass-spectrometry and protein-activity assays each have their strengths and weaknesses. Recent advances continue to improve the usefulness of these assays for systems biology.

  • Single-cell measurements are another important element in developing accurate models of biochemical events. Because only few signals can be monitored at the single-cell level, these data are most effective when combined with population-level biochemical assays.

  • Appropriate data validation and normalization techniques are crucial for constructing consistent data sets to inform modelling approaches. Several methods of data scaling can be used to highlight different features of a data set.

Abstract

Systems biology, particularly of mammalian cells, is data starved. However, technologies are now in place to obtain rich data, in a form suitable for model construction and validation, that describes the activities, states and locations of cell-signalling molecules. The key is to use several measurement technologies simultaneously and, recognizing each of their limits, to assemble a self-consistent compendium of systematic data.

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Figure 1: The scope of systems biology.
Figure 2: Merits of different assay technologies.

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Acknowledgements

This work was funded by a systems biology centre grant from the National Institutes of Health.

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Authors and Affiliations

  1. Department of Biological Engineering, Center for Cell Decision Processes, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    John G. Albeck, Forest M. White, Peter K. Sorger, Douglas A. Lauffenburger & Suzanne Gaudet

  2. Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, 02115, Massachusetts, USA

    John G. Albeck, Peter K. Sorger & Suzanne Gaudet

  3. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Gavin MacBeath

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Correspondence to Douglas A. Lauffenburger.

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Glossary

Systematic set

A data set in which all data are collected from the same experimental system in such a way that all data can be directly compared, regardless of when measurements were made.

Signal

Any type of biomolecule that transfers information in a signalling network.

Perturbation

Any experimental condition that is applied to a cell that causes a shift in the cell's behaviour away from the basal state. This includes extracellular stimulation by physiological ligands, inhibition of protein activities by small molecule inhibitors, or alterations in protein-expression levels by RNA interference or overexpression.

Immunoblot

Also known as a western blot. Following gel-based separation by mass, charge or both, proteins are transferred to a membrane and probed with target-specific antibodies.

Enzyme-linked-immunosorbent assay

(ELISA). ELISAs involve adsorbing or coupling capture antibodies to a 96-well plate. Following protein capture, a target protein is detected, either directly (if it was labelled in the sample) or indirectly, through a labelled detection antibody.

Flow cytometry

A method in which fluorescence-intensity data are recorded from particles in solution as they flow past a detector.

Protein profiling

A method that assesses the expression level of a large set of proteins in a specific tissue or cell type. It is analogous to transcriptional profiling by DNA microarrays.

Protein-interaction microarray

A protein microarray that is used to assay protein interactions. In such arrays, the capture reagents are purified proteins or protein domains, and the analyte solution contains a potential binding partner. Detection strategies are the same as in antibody microarrays (direct labelling or sandwich).

Substrate-protein microarray

A protein microarray that is used to identify substrates of enzymes, such as kinases. In this format, the array consists of potential substrates, and the analyte contains a purified enzyme. Modification of the substrates on the array (for example, phosphorylation) by the analyte is detected by radiolabel incorporation or other labelling strategies.

Microfluidic device

A device for fluid handling in which the smallest dimensions of the features (channels, valves and so on) are on the scale of a few to a few hundred micrometers.

Stable-isotope labelling with amino acids in culture

(SILAC). This method labels proteins from different samples with heavy atoms, yielding mass differences of several Daltons between the same peptide from different samples.

Isobaric tags for relative and absolute quantification

(iTRAQ). iTRAQ labels are initially isobaric, ensuring that the same peptides from different samples behave identically in the full mass spectrum (MS) mode, but they fragment to generate marker ions that differ by a single Dalton in tandem MS mode during peptide identification.

Marker ion

An ion that carries the isotope label in the breakdown of a peptide during tandem mass spectrometry analysis.

Chemosensor

In the context of kinase assays, a chemosensor is a substrate peptide that contains the non-natural amino acid Sox, which displays chelation-enhanced fluorescence when the peptide is phosphorylated.

Activity-based protein profiling

(ABPP). A method that uses reactive probes carrying a label that will covalently bind specifically to active enzymes of a certain class. The label is often a fluorophore, enabling visualization and quantification of coupled enzymes on gels, antibody microarrays or in cells. Recently, reactive probes have been labelled with an affinity tag for capture of the coupled enzymes, quantification and identification by mass spectrometry.

Image cytometry

A method that uses microscope optics to collect low-resolution data from cells that are adhered to a slide.

Bayesian network inference

A statistical method for inferring the probable relationships between measured variables.

Data validation

The process of verifying assay accuracy.

Data normalization

The adjustment of measured values to account for possible run-to-run and day-to-day variability in the assays.

Fluorescence speckle microscopy

Speckles that form by the random association of fluorophores with macromolecular structures are tracked by live-cell imaging. The information in the dynamic behaviour of these speckles is converted into a quantitative spatio-temporal readout of cytoskeleton-polymer transport and turnover.

Orthogonal design

A method of validation in which conditions that were previously varied from experiment to experiment in the course of collecting a full data set are varied in a single experiment such that what was previously separated in time now becomes contemporaneous.

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Albeck, J., MacBeath, G., White, F. et al. Collecting and organizing systematic sets of protein data. Nat Rev Mol Cell Biol 7, 803–812 (2006). https://doi.org/10.1038/nrm2042

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