Flow field-flow fractionation: recent trends in protein analysis

Abstract

Flow field-flow fractionation (F4) is the gentlest flow-assisted separation technique for analysis of macromolecules. The use of an empty channel as separation device and of a second mobile phase flow as perpendicular field enable F4 to separate analytes under native conditions without any modification of their original structure. Because of this unique peculiarity, F4 has been shown to be ideal for "gentle" separation of biological samples, for example intact proteins and protein complexes, since its early development. Today's F4 is an appealing technique which complements most established separation techniques, for example liquid chromatography and electrophoresis. The number of applications that show the unique advantages of F4 for analysis of protein samples is constantly increasing. In particular, F4 is finding increasing application on very high-molecular-weight species such as protein oligomers, aggregates, and complexes. This review critically discusses recent literature on the application of F4 to proteins. Either stand-alone or coupled with other characterization techniques, F4 is particularly promising for quality control of protein therapeutics, characterization of amyloid proteins, lipoprotein profiling, and as a pre-MS separation step in proteomics.

Instrumentation of hollow fiber flow field flow fractionation for selective cell elution

“…Sealing a connecting T equipped with appropriate flow restrictors at that position controlled the radial flow rate. Obviously, and in contrast to a HF5 glass holder or asymmetrical flow FFF (AFFFF), it was not possible to visualize the possible focusing position [13,15,16]. However, some blind focalization processes appeared effective as previously demonstrated [21] and have been systematically applied in this report.…”

“…Nevertheless, the absence of commercial interest, the lack of a large scientific application market associated with a high level of interfacing competences linking cell biology, instrumentation, chemistry, and material sciences limit the wide spread use of cell sorting by SdFFF. However, the success of all FFF techniques using flow as an external field has positioned the FFF concept in some key positions [13] for environmental studies [14] or biotechnologies [15][16][17][18][19][20]. Expansion of FFF cell sorting methodologies by means of an adapted and simplified instrumentation, which could be easily used by unspecialized laboratories, opens a new trend.…”

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

“…In native MS, where physiological or near-physiological conditions are required, the task of separating non-denatured proteins and protein complexes at high resolution is further complicated. Conventional separation techniques compatible with native MS include size exclusion chromatography (SEC) [18], ion exchange chromatography (IEX) [19], hydrophobic interaction chromatography (HIC) [20], affinity chromatography [21], capillary isoelectric focusing (cIEF) [22, 23], native gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) [24, 25], native polyacrylamide gel electrophoresis [26], capillary zone electrophoresis (CZE) [27, 28] and flow field-flow fractionation (F4) [29, 30]. However, for many of these methods, online interfacing to a mass spectrometer is challenging or yet impossible.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…In native MS, where physiological or near-physiological conditions are required, the task of separating non-denatured proteins and protein complexes at high resolution is further complicated. Conventional separation techniques compatible with native MS include size exclusion chromatography (SEC) [302], ion exchange chromatography (IEX) [163], hydrophobic interaction chromatography (HIC) [303], affinity chromatography [304], capillary isoelectric focusing (cIEF) [305,306], native gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) [167,307], native polyacrylamide gel electrophoresis [308], capillary zone electrophoresis (CZE) [67,309] and flow field-flow fractionation (F4) [310,311]. However, for many of these methods, online interfacing to a mass spectrometer is challenging or yet impossible.…”