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Next-generation proteomics: towards an integrative view of proteome dynamics

Key Points

  • Our understanding of cellular function depends on exquisite knowledge of all of the molecular components acting in a system. Mass spectrometry (MS)-based proteomics has matured immensely in the last decade, allowing quantitative system-wide analysis of the proteome, including post-translational modifications (PTMs), protein–protein interactions and cellular localization.

  • Quantification of the entire set of proteins expressed in a complex biological system (for example, mammalian cells) is now possible with a high sensitivity and in a reasonable amount of time.

  • With the availability of genomic information, the massive capacity for peptide identification by MS is being used to annotate gene sequences and to find new protein-coding genes and splicing variants.

  • In combination with new approaches to isolate specific PTMs, MS-based studies are revealing a much higher order of proteome complexity in which most proteins are modified by several PTMs that crosstalk in intricate mechanisms to regulate protein function.

  • Protein affinity strategies allow purification of candidate proteins and their interacting partners, which are subsequently identified by MS. These studies describe, with a high degree of detail, dynamic and context-specific protein–protein interaction networks and protein complexes.

  • The improvements in sensitivity, robustness and high-throughput of MS-based proteomics now permits applications in the clinical field, including the possibility of discovering disease-related biomarkers and screening molecular targets of candidate drugs.


Next-generation sequencing allows the analysis of genomes, including those representing disease states. However, the causes of most disorders are multifactorial, and systems-level approaches, including the analysis of proteomes, are required for a more comprehensive understanding. The proteome is extremely multifaceted owing to splicing and protein modifications, and this is further amplified by the interconnectivity of proteins into complexes and signalling networks that are highly divergent in time and space. Proteome analysis heavily relies on mass spectrometry (MS). MS-based proteomics is starting to mature and to deliver through a combination of developments in instrumentation, sample preparation and computational analysis. Here we describe this emerging next generation of proteomics and highlight recent applications.

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Figure 1: The diverse and dynamic mechanisms of proteome regulation provide a higher order of complexity to the human genome.
Figure 2: Generalized mass-spectrometry-based proteomics workflow.
Figure 3: Scenarios for proteome expression profiling.
Figure 4: Large-scale mass spectrometry data sets of post-translational modifications (PTMs) allow analysis of PTM crosstalk.
Figure 5: Generalized workflow for the identification, validation and stratification of protein-based biomarker signatures.


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This work was supported by the Netherlands Proteomics Centre, by the PRIME-XS project (grant number 262067, funded by the European Union 7th Framework Program) and by the Netherlands Organization for Scientific Research (NWO)-supported large-scale proteomics facility Proteins@Work (project 184.032.201).

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Correspondence to Albert J. R. Heck.

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Liquid chromatography coupled to mass spectrometry

(LC–MS). High-performance liquid chromatography is coupled to mass spectrometry to separate the peptide mixture in liquid phase on the basis of hydrophobic interactions with the C18 stationary phase of the chromatography column (C18 refers to the length of the alkyl chains that decorate the chromatographic beads).

Tandem mass spectrometry

(MS/MS). A type of mass spectrometry in which ions are selectively isolated and then fragmented. The mass-to-charge ratio of each molecular fragment is measured and used for structural characterization.

Selected reaction monitoring

(SRM). Protein quantification obtained by monitoring the specific combination of precursor and fragment ion mass-to-charge ratios of several selected peptides per protein.

Collision-induced dissociation

(CID). Fragmentation of molecular ions in the collision cell through increasing the molecules, kinetic energy followed by collisions with neutral molecules (often helium, nitrogen or argon).


Increments of 25 Da are isolated across the mass range of interest, and all ions within the mass window are simultaneously fragmented. The resulting fragments are analysed at a high resolution and afterwards are matched to a peptide fragment library.

Isobaric chemical labels

Chemical labels used in mass spectrometry that have identical molecular mass but that fragment during tandem mass spectrometry into reporter ions of different masses.

Ultra-high-performance liquid chromatography

(UHPLC). High-performance liquid chromatography carried out under extended pressure regimes (typically up to a 1,000 bar), allowing the use of smaller stationary phase particle sizes, increasing interaction volumes and thus separation power.


The use of proteomics data, which are often derived from mass spectrometry analysis, to improve gene annotations.

Induced pluripotent stem cells

(iPSCs). Somatic cells that have been reprogrammed to a pluripotent state, which is highly similar to that of embryonic stem cells, through ectopic expression of four transcription factors (namely, OCT4, SOX2, MYC and KLF4).

Embryonic stem cells

(ESCs). Pluripotent cells that can be derived from the inner cell mass of the blastocyst-stage embryo.

Electron transfer dissociation

(ETD). Fragmentation of molecular cations by the transferal of an electron.

Immobilized metal ion affinity chromatography

(IMAC). A metal ion with an affinity for analytes to be enriched (often phosphopeptides) is fixed to an insoluble matrix and serves as the adsorption centre, allowing complexation.

Basophilic kinases

Kinases that have a preference for basic amino acids in the sequence motifs of their substrates.


A carbohydrate-binding protein that is involved in various biological recognition phenomena.

Hydrophilic interaction liquid chromatography

(HILIC). Normal-phase chromatography with water-miscible mobile phases to separate hydrophilic compounds, such as proteins and peptides. Typically, the order of elution is the opposite of that obtained with reversed-phase chromatography.

Filter-aided sample preparation

(FASP). Generation of tryptic peptides from crude lysates for liquid chromatography coupled to mass spectrometry (LC–MS) analysis within a filtration device, allowing analysis of detergent lysed cells and tissues.

Tandem affinity purification

(TAP). A process in which a protein is carboxy-terminally tagged with a peptide containing a calmodulin-binding peptide, a TEV protease cleavage site and protein A. The protein is first purified using immunoglobulin-G-coated beads that bind protein A. The protein fusion is then cleaved from the gene of interest by the TEV protease and purified.

AQUA peptides

A precisely known amount of a synthetic tryptic peptide, which corresponds to a peptide of interest in the sample, with one stable-isotope-labelled amino acid that is used to determine absolute protein amounts.

Fluorescence-activated cell sorting

(FACS). A method in which dissociated and individual living cells are sorted in a liquid stream according to the intensity of fluorescence that they emit as they pass through a laser beam. Sources of fluorescence include labelled antibodies that allow cell sorting on the basis of the expression of cell-surface molecules.


Multicellular structures that resemble organs in architecture and function.

Mass cytometry

Cells bound to antibody–isotope conjugates are sprayed as single-cell droplets into an inductively coupled plasma mass spectrometer, creating a quantifiable response profile.

Ribosome profiling

Qualitative and quantitative sequencing of the RNA attached to ribosomes as a signature of genes that are expressed.

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Altelaar, A., Munoz, J. & Heck, A. Next-generation proteomics: towards an integrative view of proteome dynamics. Nat Rev Genet 14, 35–48 (2013).

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