A Multiplexed Approach for the Determination of Intact Protein Mass, Dimension and Oligomerization Using Nanoelectrospray Ion Mobility Spectrometry (GEMMA), LTQ-Orbitrap MS, MALLS and QELS

摘要

1. nES-IMS (or GEMMA) and nESI-MS (Orbitrap) requires considerably less amount of material and is faster per measured sample (by a factor of approx15 when the time for SEC column elution and regeneration is taken into account). 2. Because of short analysis times, GEMMA is better suited for large sample set screening and could make an excellent front-end to a proteomics intact protein analysis flow (if coupled on-line to a high resolution mass spectrometer Orbitrap). 3. Quaternary non-covalent protein structures "survive" gas-phase traveling in GEMMA (e.g. SecA is a dimer) whereas in Orbitrap only a minor fraction of them is finally detected. 4. GEMMA "shrinks" protein diameter by approximately 10percent, compared MALLS and QELS. The same is most probably true for the other gas-phase analysis, namely nES-MS. 5. Size and Molecular Mass measurements are similar between the compared methods. 6. Disadvantage of GEMMA and nESI-MS (Orbitrap) is that it can not be used to measure high concentration levels because artifact oligomers tend to form. 7. nESI-Orbitrap MS determines with high accuracy the protein mass while is able to identify possible Post-translational modifications, truncations and mutations which can not be obtain/predicted by the theoretical proteome of any given organism/cell. Therefore it is a necessary complementary technique to GEMMA.