Reproducibility of LC-MS-based protein identification

Traditional analysis of liquid chromatography-mass spectrometry (LC-MS) data, typically performed by re-viewing chromatograms and the corresponding mass spectra, is both time-consuming and difficult. Detailed data analysis is therefore often omitted in proteomics applications. When analysing multiple proteomics sam-ples, it is usually only the final list of identified proteins that is reviewed. Thismay lead to unnecessarily complex or even contradictory results because the content of the list of identified proteins depends heavily on the conditions for triggering the collection of tandem mass spectra. Small changes in the signal intensity of a peptide in different LC-MS experiments can lead to the collection of a tandemmass spectrum in one experiment but not in another. Also, the quality of the tandem mass spectrom-etry experiments can vary, leading to successful identi-fication in some cases but not in others. Using a novel image analysis approach, it is possible to achieve repeat analysis with a very high reproducibility by matching peptides across different LC-MS experiments using the retention time and parent mass over charge (m/z). It is also easy to confirm the final result visually. This ap-proach has been investigated by using tryptic digests of integral membrane proteins from organelle-enriched fractions from Arabidopsis thaliana and it has been demonstrated that very highly reproducible, consistent, and reliable LC-MS data interpretation can be made. Key words: DeCyderTM MS, differential expression analysis, LC-MS, reproducibility, reversed phase chromatography, nano LC, tandem mass spectrometry