Журнал протеомики и энзимологии

Evaluation of Tryptic Peptides from Neisseria meningitidis Outer Membrane Proteins PorA and PorB Digestion, Peptides

Gail Whiting, Clive Metcalfe, Adrian F Bristow and Jun X Wheeler

Abstract

Peptide-based protein quantitation using LC-MS/MS is a valuable method for the measurement of specific proteins in complex biological samples. To ensure accurate measurement of the target protein the signature peptides must be true stoichiometric representatives of the amount of protein in the sample. We report here the results of our evaluation of candidate signature peptides for IDMS quantitation of Neisseria meningitidis outer membrane proteins PorA and PorB. We observed considerable differences in the quantitation of the individual peptides and determined that the digestion protocol used was crucial to the accuracy of protein quantitation. A rapid rate of cleavage was essential to produce high quality data for the quantitation of PorA and PorB. This report also highlights the need for signature peptides and their isotopically labelled form, i.e. the internal standards, to be stable throughout digestion. However, we did observe that biased quantitation likely due to peptide instability could be mitigated by concurrent addition of the labelled internal standard peptide. Furthermore we observed that sample matrices have differing effects on specific peptide cleavage during protein digestion. In conclusion, the selection of stable signature peptides and the optimisation of the digestion conditions are not trivial considerations but are essential for accurate protein quantitation.