Hollow-fiber flow field-flow fractionation: a gentle separation method for mass spectrometry of native proteins.
Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.
Reschiglian
Pierluigi
P
Dept. of Chemistry "G. Ciamician", University of Bologna, Via Selmi 2, 40126 Bologna, Italy. pierluig.reschiglian@unibo.it
Zattoni
Andrea
A
Roda
Barbara
B
Roda
Aldo
A
Parisi
Daniela
D
Moon
Myeong-Hee
MH
Min
Byung-Ryul
BR
D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D031521:Fractionation, Field Flow / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods
D011506:Proteins