Label free quantitation approach

The quantification of protein between two physiological states can be performed, as seen previously, using protein gel staining or mass-spectrometry-based methods. These methods include differential stable isotope labeling introduced metabolically, enzymatically or even by spiked synthetic peptide standards. All these different methods rely on the addition of a chemical compound on the protein or peptide. On the opposite, label free quantification allows correlating the mass spectrometric signal of intact peptides with protein quantification directly, without any use of external chemical modification (57).

Different label free quantitation approaches have been reported so far. These approaches include replicate protocol, exponentially modified Protein Abundance Index (emPAI) and average method. The replicate protocol is based on an integrated algorithm that automatically detects and quantifies large numbers of peptide peaks aligned according to their m/z ratio and their elution time (58). These peaks are matched across many different datasets.

This approach allows quantifying a large variety of peptides and the method relies on linearity of signal compared to molecular concentration and on reproducibility of sample processing (59).

The emPAI approach is based on the estimation of absolute protein content in a complex mixture using the protein abundance index (PAI) (number of observed peptides divided by the number of observable peptide per protein) (60). This PAI value shows a linear relationship with the logarithm of protein concentration and can be used as a quantitative tool in proteomic studies. Another label-free quantitation method has been reported by Silva (et al.) and is based on the rule that the average peak height for the three most intense tryptic peptides per mole of protein is constant within a variation of ±10%. By adding an internal standard this relationship can give an absolute quantitation of the protein tested by calculating a universal signal response factor (counts/mol) applicable to all the proteins tested in their study (61).

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