Mass Spectrometry: Analysis of Two‐Dimensional Protein Gels

Abstract

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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References

Anderson NL and Anderson NG (2002) The human plasma proteome: history, character, and diagnostic prospects. Molecular and Cellular Proteomics 1 (11): 845–867.

Berth M , Moser FM , Kolbe M and Bernhardt J (2007) The state of the art in the analysis of two‐dimensional gel electrophoresis images. Applied Microbiology and Biotechnology 76 (6): 1223–1243.

Bjellqvist B , Ek K , Righetti PG , et al. (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. Journal of Biochemical and Biophysical Methods 6 (4): 317–339.

Butt RH and Coorssen JR (2013) Coomassie blue as a near‐infrared fluorescent stain: a systematic comparison with Sypro Ruby for in‐gel protein detection. Molecular and Cellular Proteomics 12 (12): 3834–3850.

Cravatt BF , Simon GM and Yates JR 3rd. (2007) The biological impact of mass‐spectrometry‐based proteomics. Nature 450 (7172): 991–1000.

Eng JK , Searle BC , Clauser KR and Tabb DL (2011) A face in the crowd: recognizing peptides through database search. Molecular and Cellular Proteomics 10 (11): R111.009522. DOI: 10.1074/mcp.R111.009522.

Fenn JB , Mann M , Meng CK , et al. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246 (4926): 64–71.

Fernandez M and Albar JP (2012) 2D DIGE for the analysis of RAMOS cells subproteomes. Methods in Molecular Biology 854: 239–252 (In: Cramer R and Westermeier R (eds) Difference Gel Electrophoresis (DIGE): Methods and Protocols, chapter 17).

Gelpí E (2009) From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part II 1985–2000. Journal of Mass Spectrometry 44 (8): 1137–1161.

Hecker M , Antelmann H , Büttner K and Bernhardt J (2008) Gel‐based proteomics of Gram‐positive bacteria: a powerful tool to address physiological questions. Proteomics 8 (23–24): 4958–4975.

Hillenkamp F and Karas M (1990) Mass spectrometry of peptides and proteins by matrix‐assisted ultraviolet laser desorption/ionization. Methods in Enzymology 193: 280–295.

Kolin A (1954) Separation and concentration of proteins in a pH field combined with an electric field. Journal of Chemical Physics 22: 1628–1629.

ÓFarrell PH (1975) High‐resolution two‐dimensional electrophoresis of proteins. Journal of Biological Chemistry 250: 4007–4021.

Pappin DJ (1997) Peptide mass fingerprinting using MALDI‐TOF mass spectrometry. Methods in Molecular Biology 64: 165–173.

Picotti P , Bodenmiller B , Mueller LN , et al. (2009) Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138 (4): 795–806.

Riederer BM (2008) Non‐covalent and covalent protein labeling in two‐dimensional gel electrophoresis. Journal of Proteomics 71 (2): 231–244.

Ripoll VM , Lambrianides A , Pierangeli SS , et al. (2014) Changes in regulation of human monocyte proteins in response to IgG from patients with antiphospholipid syndrome. Blood 124 (25): 3808–3816.

Suarez G , Romero‐Gallo J , Piazuelo MB et al. (2015) Modification of Helicobacter pylori peptidoglycan enhances NOD1 activation and promotes cancer of the stomach. Cancer Research Mar 2. [Epub ahead of print] PubMed PMID: 25732381.

Suzuki Y , Takagi N , Sano T and Chimuro T (2013) Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system. Electrophoresis 34 (17): 2464–2472.

Ünlü M , Morgan ME and Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18 (11): 2071–2077.

Vandenbogaert M , Hourdel V , Jardin‐Mathé O , et al. (2012) Automated phosphopeptide identification using multiple MS/MS fragmentation modes. Journal of Proteome Research 11 (12): 5695–5703.

Wilkins M (2009) Proteomics data mining. Expert Review of Proteomics 6 (6): 599–603.

Further Reading

Cao Z , Tang HY , Wang H , et al. (2012) Systematic comparison of fractionation methods for in‐depth analysis of plasma proteomes. Journal of Proteome Research 11 (6): 3090–3100.

Domon B and Aebersold R (2006) Mass spectrometry and protein analysis. Science 312 (5771): 212–217.

Duncan MW , Aebersold R and Caprioli RM (2010) The pros and cons of peptide‐centric proteomics. Nature Biotechnology 28 (7): 659–664.

Gruhler A , Olsen JV , Mohammed S , et al. (2005) Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Molecular and Cellular Proteomics 4 (3): 310–327.

Hanash SM , Pitteri SJ and Faca VM (2008) Mining the plasma proteome for cancer biomarkers. Nature 452 (7187): 571–579.

Hernández H and Robinson CV (2007) Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nature Protocols 2 (3): 715–726.

Link AJ , Eng J , Schieltz DM , et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology 17 (7): 676–682.

Olsen JV and Mann M (2013) Status of large‐scale analysis of post‐translational modifications by mass spectrometry. Molecular and Cellular Proteomics 12 (12): 3444–3452.

Patel VJ , Thalassinos K , Slade SE , et al. (2009) A comparison of labeling and label‐free mass spectrometry‐based proteomics approaches. Journal of Proteome Research 8 (7): 3752–3759.

Tran JC , Zamdborg L , Ahlf DR , et al. (2011) Mapping intact protein isoforms in discovery mode using top‐down proteomics. Nature 480 (7376): 254–258.

Wang X and Zhang B (2014) Integrating genomic, transcriptomic, and interactome data to improve Peptide and protein identification in shotgun proteomics. Journal of Proteome Research 13 (6): 2715–2723.

Witze ES , Old WM , Resing KA and Ahn NG (2007) Mapping protein post‐translational modifications with mass spectrometry. Nature Methods 4 (10): 798–806.

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