Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis

Improved CE(SDS)‐CZE‐MS method utilizing an 8‐port nanoliter valve

“…For this reason, the pre‐ and postsample zones were positioned prior to sample transfer. Applying the previous SDS‐removal strategy performed with the 4‐port valve [6, 7], with methanol as presample zone and CTAB solution in methanol: water 1:1 as postsample zone, the current was instable or even broke down completely. To understand these current instabilities, offline solubility experiments were performed.…”

“…To evaluate the overall performance in a two‐dimensional approach, CE(SDS)‐CZE‐MS analysis were performed with the presented 8‐port‐valve and optimized decomplexation in terms of peak intensity and area. The obtained results with the optimized decomplexation strategy regarding peak intensity were compared to partly published CE(SDS)‐CZE‐MS measurements of the NIST mAb LC and HC (c = 1 mg/mL) performed with the 4‐port valve and its corresponding methanol decomplexation strategy [6]. The comparison of the runs with both CE(SDS)‐CZE‐MS systems is demonstrated by the EIEs (sum of the eight most abundant m/z ) with the mean intensity ( n = 3) for LC (Fig.…”

“…An 8‐port‐valve was designed consisting of eight symmetrically positioned ports on the stator and four symmetrically positioned sample loops (4 × 20 nL) on the rotor. The stator is made of polyether ether ketone (PEEK) and the rotor of Valcon E (polyaryletherketone (PAEK)/PTFE‐based composite) as described before [6]. The valve was custom made by VICI AG International (Schenkon, Switzerland; part number: C5M‐4354‐CU1), equipped with 360 μm fittings and a multiposition electric microactuator.…”

“…None of these approaches was utilized for the CE(SDS) application so far. Hence, we recently introduced a 2D capillary electrophoretic separation system utilizing a 4‐port nanoliter valve [5] for online mass spectrometric detection of CE(SDS)‐separated peaks [6]. The generic CE(SDS) separation dimension, as first dimension ( 1 D), is hyphenated via the 4‐port nanoliter valve with a MS‐compatible CZE method as second dimension ( 2 D), for separating interfering substances from the peak of interest prior to introduction into the MS. One crucial part of this CE(SDS)‐CZE‐MS approach is the decomplexation of the SDS‐protein complex in the 2 D. An in‐capillary decomplexation strategy was developed in our group, by testing several cationic compounds, revealing that the cationic surfactant CTAB and SDS form a more stable complex then SDS with proteins, releasing the free protein [7].…”

“…The generic CE(SDS) separation dimension, as first dimension ( 1 D), is hyphenated via the 4‐port nanoliter valve with a MS‐compatible CZE method as second dimension ( 2 D), for separating interfering substances from the peak of interest prior to introduction into the MS. One crucial part of this CE(SDS)‐CZE‐MS approach is the decomplexation of the SDS‐protein complex in the 2 D. An in‐capillary decomplexation strategy was developed in our group, by testing several cationic compounds, revealing that the cationic surfactant CTAB and SDS form a more stable complex then SDS with proteins, releasing the free protein [7]. Thus, in relation to the SDS‐protein sample, that is transferred via sample loop from the 1 D to the 2 D, a presample zone of solvent (methanol) and a postsample zone of CTAB is utilized for decomplexation in the 2 D. Therefore, the zones were hydrodynamicaly injected via the second CZE dimension and positioned with the help of C 4 D. In this way, several mAb fragments and soybean proteins separated by CE(SDS) were characterized by MS [6]. However, robustness was limited due to frequent current leakage and breakdown, most probably caused by insufficient distance between the separation dimensions and inaccurate positioning of the decomplexation zones in the 2 D.…”