Fig. 6.

Principle of LOPIT and label-free quantification. The LOPIT technology is based on the notion that biochemical purification of an organelle never results in an absolutely homogeneous preparation. Instead, proteins that are specifically associated with the organelle of interest (green dots) become enriched upon purification (top layer; a), and contaminants (red dots), although still present in the enriched fraction a, become depleted. The LOPIT technology uses labeling of peptides with stable isotopes such as ICAT or iTRAQ (right-hand side of the figure). For this, peptides from the enriched (a) and depleted (b and c) samples are labeled with reagents of different masses, mixed together, and processed by liquid chromatography and mass spectrometry. The isotope labeling allows relative quantitation of the protein abundance in the enriched and depleted fractions simultaneously (a, b, and c). For the label-free quantitative approach (left-hand side of the figure), peptides from the enriched (a) and depleted (b and c) fractions are processed by liquid chromatography and mass spectrometry separately without labeling or mixing. In this case, the protein abundance in each fraction is quantified based on the number of observed peptides divided by the number of observable peptides (protein abundance index), the peptide peak intensity (protein correlation profiling), or the spectral count (number of identified spectra for each protein).