Mass Spectrometry: Analysis of Two‐Dimensional Protein Gels


Introduced two decades ago, two‐dimensional gel electrophoresis (2‐DE) in combination with mass spectrometry has since been a powerful tool to analyse and to describe the enormous complexity hidden in the proteomes. Massively used by the academic community in the last two decades, it has lost its privileged status in favour of other high‐throughput approaches, such as liquid chromatography coupled to mass spectrometry (LC‐MS). Nevertheless, 2‐DE is still a useful tool to describe both in qualitative and quantitative terms the complexity of the proteome, offering some advantages over ‘peptide‐centric’ approaches such as LC‐MS and a more ‘realistic’ view of common post‐translational processes that modify both the structure and function of proteins, including alternative splicing processes and common post‐translational modifications such as phosphorylation and glycosylation. In addition, continuous improvements in gel imaging software have greatly improved the utility of 2‐DE for quantitative experiments.

Key Concepts

  • Analysis of complex proteomes demand the use of high‐resolution separation techniques.
  • Immobilised pH gradients increase the resolution power of two‐dimensional gel electrophoresis (2‐DE) while reducing experimental variability.
  • 2‐DE gel‐based protein analysis has lost predominance in favour of peptide‐centric experimental approaches.
  • 2‐DE is a powerful experimental approach to compare complex proteomes quantitatively.
  • 2‐D DIGE significantly reduces artefactual gel‐to‐gel variability.
  • Development of soft‐ionisation techniques made it possible to analyse peptides and proteins by means of mass spectrometry.
  • MALDI TOF TOF mass spectrometers are best suited for the analysis of 2‐DE protein spots.
  • High performance liquid chromatography can be directly coupled to ESI‐based mass spectrometers.

Keywords: proteome; proteomics; two‐dimensional gel electrophoresis; immobilised pH gradients; electrospray ionisation; matrix‐assisted laser desorption/ionisation; mass spectrometry; high‐throughput proteomics

Figure 1. Schema depicting the different parts that build current mass spectrometers. Those elements that are most frequently used in proteomics‐oriented mass spectrometers are shown in bold. HPLC, high‐pressure liquid chromatography; GC, gas chromatography; MALDI, matrix‐assisted laser desorption/ionisation; ESI, electrospray ionisation; FAB, fast atom bombardment; LSIMS, liquid secondary ion mass spectrometry; EI/CI, electronic impact/chemical ionisation; TOF, time‐of‐flight; FTMS, Fourier transform mass spectrometry.
Figure 2. MS spectrum and tandem MS spectra corresponding to a MALDI‐TOF TOF mass spectrometry analysis of the tryptic peptide mixture obtained after in‐gel digestion of a protein spot. Tandem MS spectra of three of the most intense signals found in the MS spectrum as well as the corresponding peptide identification are shown.
Figure 3. Strategy for high‐throughput identification of protein spots obtained from two‐dimensional gels. After software‐aided image analysis, protein spots of interest are excised (manually or using automatic spot‐pickers) and in‐gel digested with trypsin. Automatic robots reduce the presence of undesired contaminants (e.g. keratins) in the sample. Resulting tryptic digests are screened by a combination of matrix‐assisted laser desorption/ionisation (MALDI) and high‐resolution time‐of‐flight (TOF) mass spectrometry (MS) for protein identification. MS and tandem MS spectra are used to search against specific protein databases. Peptide‐mass fingerprinting combined with tandem MS analysis is sufficient to identify the majority of spots.


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Paradela, Alberto(Jul 2015) Mass Spectrometry: Analysis of Two‐Dimensional Protein Gels. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0003110.pub2]