Preparation Artefacts in Proteomics: Technological Issues


In addition to taking into account the more than 200 endogenously produced posttranslational modifications, a detailed analysis of proteins separated by two‐dimensional electrophoresis must consider other modifications that a protein can experience during the various steps of its separation.

Keywords: proteomics; protein alkylation; iodoacetamide; acrylamide; 4‐vinylpyridine; electrophoresis

Figure 1.

Two‐dimensional map of (a) reduced but nonalkylated and (b) reduced and alkylated globin chains. Note in (b) the complete removal of all higher‐order oligomers (except for traces of dimers) and the detection of minor components of red blood cell lysates, such as carbonic anhydrase and thioredoxin peroxidase, which would have been hidden under the string of dimers in nonalkylated samples. (After Herbert et al..)

Figure 2.

Reaction of the –SH groups of bovine α‐lactalbumin (LCA) with (a) dimethyl acrylamide (DMA) or (b) 4‐vinylpyridine (4‐VP) in the presence of2% Triton X‐100.

Figure 3.

MALDI‐TOF mass spectra of human albumin: (a) before alkylation; (b, c) after incubation with iodoacetamide (IAA) for 1 h (b) and 6 h (c); and (d) after incubation with 4‐vinylpyridine (4‐VP) for 1 h.



Bordini E, Hamdan M and Righetti PG (1999) Matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry for monitoring alkylation of β‐lactoglobulin B exposed to a series of N‐substituted acrylamide monomers. Rapid Communications in Mass Spectrometry 13: 2209–2215.

Bordini E, Hamdan M and Righetti PG (2000) Alkylation power of free Immobiline chemicals towards proteins in isoelectric focusing and 2D maps as explored by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. Electrophoresis 21: 2911–2918.

Galvani M, Bordini E, Piubelli C and Hamdan M (2000a) Effect of the experimental condition on the analysis of SDS‐PAGE separated proteins by matrix assisted laser desorption/ionisation mass spectrometry. Rapid Communications in Mass Spectrometry 14: 18–25.

Galvani M, Hamdan M and Righetti PG (2000b) Investigating the reaction of a number of gel electrophoresis crosslinkers with β‐lactoglobulin by matrix‐assisted laser desorption/ionisation mass spectrometry. Electrophoresis 21: 3684–3692.

Galvani M, Hamdan M, Herbert B and Righetti PG (2001) Alkylation kinetics of proteins in preparation for two‐ dimensional maps/MALDI‐TOF mass spectrometry investigation. Electrophoresis 22: 2058–2065.

Gillespie PG and Gillespie SKH (1997) Improved electrophoresis and transfer of picogram amounts of protein with hemoglobin. Analytical Biochemistry 246: 239–245.

Herbert B, Galvani M, Hamdan M, et al. (2001) Reduction and alkylation of proteins in preparation of two dimensional map analysis: why, when and how? Electrophoresis 22: 2046–2057.

Further Reading

Godovac‐Zimmermann J and Brown LR (2001) Perspectives for mass spectrometry and functional proteomics. Mass Spectrometry Reviews 20: 1–57.

Hamdan M, Galvani M and Righetti PG (2001) Monitoring 2D gel‐induced modifications of proteins by MALDI‐TOF mass spectrometry. Mass Spectrometry Reviews 20: 121–141.

Hanash SM (1998) Two‐dimensional gel electrophoresis. In: Hames BD (ed.) Gel Electrophoresis of Proteins, pp. 189–211. Oxford, UK: Oxford University Press.

Kellner R, Lottspeich F and Meyer HE (1999) Microcharacterization of Proteins. Weinheim: Wiley‐VCH.

Rabilloud T (ed.) (2000) Proteome Research: Two‐dimensional Gel Electrophoresis and Identification Methods. Berlin: Springer.

Righetti PG (1983) Isoelectric Focusing: Theory, Methodology and Applications. Amsterdam: Elsevier.

Righetti PG (1990) Immobilized pH Gradients: Theory and Methodology. Amsterdam: Elsevier.

Righetti PG, Stoyanov A and Zhukov M (2001) The Proteome Revisited: Theory and Practice of All Relevant Electrophoretic Steps. Amsterdam: Elsevier.

Westermeier R (1997) Electrophoresis in Practice. Weinheim: VCH.

Wilkins MR, Williams KL, Appel RD and Hochstrasser DF (eds.) (1997) Proteome Research: New Frontiers in Functional Genomics. Berlin: Springer.

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Righetti, Pier Giorgio, and Hamdan, Mahmoud(Jan 2006) Preparation Artefacts in Proteomics: Technological Issues. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0006192]