Heart‐cut nano‐LC–CZE–MS for the characterization of proteins on the intact level

Abstract

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed-phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well-suited geometries/dimensions. Here, a heart-cut nano-LC-CZE-MS setup was developed utilizing for the first time a mechanical 4-port valve as LC-CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart-cut transfer of individual LC peaks and subsequent CZE-MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower g/mL range were determined, which are considerably lower compared to traditional CZE-MS. In addition, this study represents the first application of an LC-CE-MS system for intact protein analysis. The nano-LC-CZE-MS system is expected to be applicable to various other analytical challenges.Additional supporting information may be found online in the Supporting Information section at the end of the article.LC and CZE are both powerful separation techniques that can adequately resolve components in various samples. Still, the demands for sensitive analysis and high-separation power are constantly increasing. One way to improve separation capabilities is to perform multidimensional analysis [1]. In this regard, LC-CE combinations are of great interest, such as reversed-phase liquid chromatography (RPLC) involving hydrophobic interactions and CZE utilizing differences in the electrophoretic mobilities of ionic substances possess nearly orthogonal separation mechanisms. Certain following challenges are needed to be faced considering the hyphenation of LC and CE technology: (i) compatibility of LC and CE conditions including flow rates, geometries, and eluent constitution; (ii) transfer of LC peaks in the low-to-middle nanoliter range; and (iii) electrical insulation required in CE.

Characterisation of a new online nanoLC-CZE-MS platform and application for the glycosylation profiling of alpha-1-acid glycoprotein

“…Peak volumes of ~ 60 nL increase the transfer efficiency to 33% (volume) and 55% (analyte). This transfer efficiency is higher than in our previous report [21] and considerably higher than typical transfer efficiencies of ~ 1% that can be achieved with flow-gating interfaces [40]. We consider this transfer efficiency of 55% (analyte) as an optimum as a further increase in transfer efficiency increases the likelihood of co-transferring adjacent peaks due to a transfer window that is too wide.…”

“…The 1 D separation was performed with either of the two nanoLC methods (300 or 100 nL/min) as described before. To couple the 1 D with the 2 D, a mechanical four-port valve with an internal 20-nL loop on the rotor was used as previously described in detail [18,21]. The detection capillary of the nanoLC was connected to one of the ports of the valve with ~ 12 cm between the UV window and valve inlet.…”

“…The low internal volumes (4-40 nL), as well as the insulating material, makes these valves promising for LC-CZE coupling. Based on a proof-of-concept study, we demonstrated the general applicability of such a valve to couple nanoLC and CZE-MS [21]. A comprehensive review and comparison of LC-CZE systems, as well as relevant applications, was recently published by Ranjbar et al [4].…”

Improved Nanoflow RPLC-CZE-MS/MS System with High Peak Capacity and Sensitivity for Nanogram Bottom-up Proteomics

“…Recently, the Neusuß group built an online RPLC-CZE-MS system using a 4-port valve as the interface for characterization of intact proteins. 17 An RPLC eluate containing proteins was selectively transferred into a sample loop integrated into the 4-port valve, followed by CZE-MS analysis. Although some successful examples were published, some challenges remain for the online RPLC-CZE-MS. First, the separation power of RPLC and CZE cannot be fully used because we need to balance the two dimensions for online operation.…”

“…Several research groups have developed online RPLC-CZE-MS systems for proteomics. − The Ramsey group carried out the connection of RPLC and CZE-MS using a microfluidic device. , They applied the online RPLC-CZE-MS systems in bottom-up MS characterization of standard protein digests, an antibody, and an E. coli proteome sample. Under the optimal condition, a peak capacity of over 1000 in 1 h was produced by the online system.…”