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Real and imaginary artefacts in proteome analysis via two-dimensional maps
1Polytechnic of Milano, Department of Chemistry, Giulio Natta, Materials and Engineering Chemistry, Via Mancinelli 7, Milano 20131, Italy. piergiorgio.righetti@polimi.it
Summary
Two-dimensional mapping artefacts, including deamidation and carbamylation, are often misattributed. Proper sample handling and alkylation are crucial to prevent oligomerization and desulfuration artefacts during electrophoresis.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Two-dimensional mapping is a key proteomics technique.
- Artefacts in 2D mapping can lead to misinterpretation of results.
- Previous studies have focused on deamidation and carbamylation as major artefact sources.
Purpose of the Study:
- To review and clarify artefacts in two-dimensional mapping.
- To identify and explain previously underestimated artefacts.
- To provide recommendations for artefact prevention.
Main Methods:
- Literature review of artefacts in gel-based isoelectric focusing and 2D mapping.
- Analysis of protein modification mechanisms during electrophoresis.
- Evaluation of sample preparation and treatment protocols.
Main Results:
- Deamidation and carbamylation are unlikely artefacts in properly handled samples.
- Homo- and hetero-oligomer formation is a significant artefact in unalkylated samples, especially at alkaline pH.
- Desulfuration of cysteine residues leads to protein degradation during prolonged electrophoresis.
- Alkylation of samples prevents oligomerization and desulfuration, preserving sample integrity.
Conclusions:
- Proper alkylation is essential to prevent major artefacts like oligomerization and desulfuration in 2D mapping.
- Focusing on sample integrity through correct handling and alkylation is key for accurate proteomic analysis.
- Understanding and mitigating these artefacts improves the reliability of 2D mapping results.