Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection

Abstract

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low-level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Páger et al. developed a CIEF‐MS method applying narrow range ampholytes by sequential injection . They showed that this sequential injection protocol enabled the detection of CIEF‐separated proteins.…”

The effect of pH adjusted electrolytes on capillary isoelectric focusing assessed by high‐resolution dynamic computer simulation

“…Such a configuration permits focusing to be executed under concomitant mobilization effected by EOF [11][12][13][14][15][16][17][18][19], application of hydrodynamic flow, electrophoretic transport when the leading electrode solution contains a counter ion other than H 3 O + and OH − [18,[20][21][22][23][24][25] or with combinations of these principles [18,23]. These approaches are reported to be attractive for hyphenation of CIEF with ESI-MS via use of volatile electrolytes such as formic acid and ammonium hydroxide [17,19,26] and for on-target fraction collection for offline coupling of CIEF and MALDI-MS [23]. In this context, Kilár et al introduced a sequential injection configuration where the carrier ampholytes and samples are injected separately [16][17][18][19].…”

“…These approaches are reported to be attractive for hyphenation of CIEF with ESI-MS via use of volatile electrolytes such as formic acid and ammonium hydroxide [17,19,26] and for on-target fraction collection for offline coupling of CIEF and MALDI-MS [23]. In this context, Kilár et al introduced a sequential injection configuration where the carrier ampholytes and samples are injected separately [16][17][18][19].…”

“…Alternatively, CIEF can be run via application of the sample and carrier ampholytes within part of the column on the inlet side while the rest of the column is occupied by one of the electrode solutions, i.e., the catholyte or the anolyte. Such a configuration permits focusing to be executed under concomitant mobilization effected by EOF [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ], application of hydrodynamic flow, electrophoretic transport when the leading electrode solution contains a counter ion other than H 3 O + and OH − [ 18 , 20 , 21 , 22 , 23 , 24 , 25 ] or with combinations of these principles [ 18 , 23 ]. These approaches are reported to be attractive for hyphenation of CIEF with ESI‐MS via use of volatile electrolytes such as formic acid and ammonium hydroxide [ 17 , 19 , 26 ] and for on‐target fraction collection for offline coupling of CIEF and MALDI–MS [ 23 ].…”

“…In this context, Kilár et al. introduced a sequential injection configuration where the carrier ampholytes and samples are injected separately [ 16 , 17 , 18 , 19 ].…”