Smart proteolytic samplers for liquid chromatography-mass spectrometry protein quantification: Assessing trypsin immobilization via divinyl sulfone or periodate functionalization

Abstract

In this report we try to answer the question if smart proteolytic samplers can be used for LC-MS based quantitative determination of proteins from paper. Production of smart proteolytic samplers is a two-stage process: In the first stage cellulose is functionalized while in the second stage trypsin is covalently bound. Two functionalization strategies, divinyl sulfone - and periodate functionalization, were compared under various conditions. The divinyl sulfone functionalization in combination with trypsin immobilization is here described for the first time. This functionalization chemistry shows to be equally successful as the periodate functionalization. Using Whatman Grade CF12 paper, it appears that smart proteolytic samplers with high tryptic activity can be produced. These have the capability to produce tryptic peptides with good linearity in buffered solutions: up to 0.998 for BSA (using HLVDEPQNLIK as signature peptide and IO₄-functionalization) and 0.990 for Cytochrome C (using MIFAGIK as signature peptide and IO₄-functionalization). The reproducibility in complex biological samples is acceptable (<20 % average RSD for divinyl sulfone functionalized paper) when internal correction is carried out. Overall, smart proteolytic samplers can be used for reliable LC-MS based determination of proteins when the correct proteotypic peptides are chosen.