Optimized two-dimensional nano-liquid chromatography tandem mass spectrometry protocols for proteomics applications: on-line or off-line coupling?

摘要

Results of both on-line and off-line modes remain comparable. The number of peptides identified in off-line is slightly lower probably because of the dispersion of peptides between diverse fractions but also because of the drying step. Even if peak capacity in off-line mode is twice less than in on-line mode, off-line configuration offers the possibility not to use the entire volume of the fractions. Indeed remaining volume can be analyzed by a complementary mass spectrometric approach with MALDI ionization (see ThPI Micro-scale Separations MS - 239 poster). Isocratic step gradient in on-line configuration allows to compensate the peak broadening induced by the non punctual injection of our system. Large volume injection is specific to biological studies. The amount of material is too low to afford any prior concentration step. It would induce non negligible sample losses and prevent any final detection. To correct this peak broadening we intend to evaluate an on-line pre-concentration step on the SCX, similar to the classical RP-HPLC one.