Separation techniques hyphenated to electrospray‐tandem mass spectrometry in proteomics: Capillary electrophoresis versus nanoliquid chromatography

Abstract

Liquid chromatography (LC) nanoelectrospray-tandem mass spectrometry (MS/MS) is a key technology for the study of proteomics, with the main benefit to the characterization of sensitive peptides from complex mixtures. Capillary electrophoresis coupled to mass spectrometry (MS) has been taken into consideration sporadically due to the highly efficient separation and ability to handle low sample amount, yet classified as being less sensitive with respect to analyte concentration. The limitation in capillary zone electrophoresis (CZE) injection volumes can be overcome by on-line solid-phase extraction (SPE). Such an on-line SPE-CZE system was explored in combination with an ion trap (IT) mass spectrometer. Thus, it was possible to inject more than 100 microL sample solution on to the CZE capillary. Concentration limits of detection as low as 100 amol/microL were demonstrated for a peptide standard. This SPE-CZE-microelectrospray ionization (ESI)-MS/MS setup was compared directly to nanoLC/nanoESI using the same sample of a tryptic digest of bovine serum albumin (BSA) as a reference standard. Measurements were made on one IT mass spectrometer with identical acquisition parameters. Both chromatography systems enabled the separation and detection of low levels of peptides from a mixture of moderate complexity, with most peptides identified using both techniques; however, specific differences were obvious. The nanoLC-MS is about five times more sensitive than the CZE-MS, yet the difference was less pronounced than expected. The CZE-MS technique showed reduced loss of peptides, especially for larger peptides (missed cleavages) and is about four times faster than the nanoLC-MS approach.

Efficient and highly reproducible capillary electrophoresis–mass spectrometry of peptides using Polybrene‐poly(vinyl sulfonate)‐coated capillaries

“…However, LC-MS of peptides is often relatively slow with typical analysis times of 0.5-2 h, particularly when complex mixtures have to be separated. CE is a powerful alternative for the analysis of peptides exhibiting a selectivity that is based on charge-to-mass ratio differences and therefore quite different from the separation mechanism of RPLC [1]. Furthermore, CE offers high separation efficiencies and speed, and requires only very low sample volumes [2].…”

Quantitation in capillary electrophoresis-mass spectrometry

“…The flow rate of the sheath liquid should be as low as possible to reduce dilution, despite the observation that the CE flow and the sheath liquid are probably not completely mixed [33]. On the other hand, the flow rate should not fall below a certain value, first of all to keep the spray stable, and, moreover, to avoid peak broadening, as illustrated by Vuorensola et al [26] for catecholamines.…”

“…Different approaches of on-line SPE preconcentration in the CE capillary, e.g., [63] have been developed. The SPE-CE-MS approach has especially been applied in the context of peptide analysis [33,[64][65][66]. The usefulness of online SPE-CE-MS for quantitative measurements has been shown by Waterval et al [67].…”

Toward high sequence coverage of proteins in human breast cancer cells using on‐line monolith‐based HPLC‐ESI‐TOF MS compared to CE MS

“…In CE-MS using a sheathless interface the electrical connection has been found to be the limiting factor where the connection needs to be redone after a couple of runs. Although CE can provide much improved resolution [38] compared to the monolithic columns in the 10 min separation interval, with the use of MS analysis sufficient resolution is obtained for distinguishing each peak. In addition, the monolithic columns provide much improved loadability compared to CE and much shorter separation times compared to packed capillary chromatography.…”